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Philippine crocodiles Crocodylus mindorensis are critically endangered due to agricultural and fishing threats that have severely fragmented their habitat and population in the Philippines. Captive management plans are important to safeguard against their extinction, but the current population in US zoos is small, and breeding is hampered by the slow growth of this species and the danger of introducing differently sized animals for breeding. There is little information regarding the sperm characteristics of crocodilians, and none for Philippine crocodiles. In this study, we sought to characterise sperm production in the male Philippine crocodile (n = 1) by performing voluntary (without sedation or restraint) collections (n = 181) over a 3.5-year period. Peak sperm production in this individual occurs from January to July, when the mean (±s.e.m.) total number of spermatozoa recovered was 10.2 × 106 ± 3.8 × 106 (n = 104), compared with 0.3 × 106 ± 0.2 × 106 (n = 71) for all other months of the year. Analysis of sperm morphology indicated that 15.9% of spermatozoa exhibited normal morphology. A bent tail was the most common abnormality (48.2%) observed. Understanding the basic reproductive biology of the male Philippine crocodile will facilitate the development of artificial reproductive technologies to improve captive propagation and genetic management of this species.

Spix’s cavy is a potentially good experimental model for research on reproductive biology and sexual development. The aim of the present study was to evaluate the ontogeny of the steroidogenic enzymes involved in testicular androgen synthesis during prenatal development. Testes were investigated on Days 25, 30, 40 and >50 of gestation. Immunohistochemistry and immunoblotting were used to establish the site and relative amount of androgenic enzymes, including 5α-reductase, cytosolic 17β-hydroxysteroid dehydrogenase (17β-HSDI) and mitochondrial microsomal 3β-hydroxysteroid dehydrogenase (3β-HSDII), throughout prenatal development. The testicular parenchyma began to organise on Day 25 of gestation, with the development of recognisable testicular cords. The mesonephros was established after Day 25 of gestation and the ducts differentiated to form the epididymis, as testicular cords were beginning to proliferate and the interstitium to organise by Day 30 of gestation, continuing thereafter. The androgen-synthesising enzymes 5α-reductase, 17β-HSDI and 3β-HSDII were evident in Leydig cells as they differentiated at all subsequent gestational ages studied. In addition, immunoblotting showed an increase in immunoreactivity for the enzymes at Days 30 and 40 of gestation (P

Assisted reproductive technologies (ARTs) have a significant role to play in reptile conservation, yet are severely lacking. Previous attempts to cryopreserve spermatozoa in the threatened lizard Varanus panoptes achieved approximately 48% motile sperm post-thaw for samples frozen immediately after collection. However, the feasibility of extended cold storage before cryopreservation has not been tested. We held V. panoptes spermatozoa at either 25°C or 4°C for 8 days, assessing sperm motility at days 1, 2, 4 and 8. Subsamples were cryopreserved on days 1 and 4 following the previously reported protocol for this species. Percentage motility decreased rapidly at 25°C, but did not decrease significantly until 4 days after collection at 4°C, with >30% motility maintained after 8 days. There was no significant difference in post-thaw motility or viability of samples cryopreserved after 1 or 4 days storage at 4°C, yielding substantial results for both parameters (mean motility 23.8% and 28.1% and mean viability 50.1% and 57.5% after 1 and 4 days respectively). We demonstrate the capacity to extend sperm viability for up to 8 days in unfrozen samples and to produce acceptable post-thaw motility in samples frozen after 4 days of storage, contributing to the development of valuable ARTs for lizards and other reptiles.

The Arunachali breed of yak is reared by the Monpa pastoral community of Arunachal Pradesh in India for their livelihood and nutritional security on highland pastures. In addition to fulfilling the basic needs of highlanders, this animal is associated with poor production potential linked to its inherent reproductive problems, including silent oestrus, seasonality, and inbreeding, making yak rearing a less-profitable venture. In the recent past, efforts have been made to improve reproductive efficiency of Arunachali yak through assisted reproductive techniques. However, a few regimens for superovulation have been tried to date with limited success in terms of superovulatory response and embryo recovery. Therefore, the present study was undertaken to evaluate the efficacy of two different doses and regimes of superovulation: Stimufol (400 and 200µg per animal) and Folligon (1500 and 1000IU per animal) in Arunachali yak. Twenty-four yaks were allotted in 4 groups (of 6 animals each) and subjected to respective superovulation treatment. Thereafter, the treatment response was assessed in terms of expression of oestrus, interval between treatment and oestrus onset, duration of oestrous, number of corpora lutea and embryos recovered, and analysed using two-way statistical analysis between treatment groups. The results (Table 1) showed a significant (P

The present study aimed to evaluate the effects of exogenous administration of melatonin to male camels on reproductive behaviour, seminal parameters, and serum concentrations of melatonin and testosterone during the non-breeding and subsequent breeding seasons. In September 2018, male camels (n=12) were randomly allocated to subcutaneous injections of either melatonin (MLT, 0.643 mg/kg; n=6) dissolved in corn oil, or plain corn oil; (control n=6). The camels were observed for behavioural signs of rut. A second dose of either MLT (1 mg/kg) or corn oil (control) was administered on 7 October 2018 subcutaneously, as camels did not show any change in behaviour until 6 October 2018, and the camels were again observed for behavioural signs of rut. Blood was collected from all camels on 1 September 2018, and then at weekly intervals until 7 January 2019. The serum was separated and stored until further assay of serum melatonin and testosterone using ELISA kits. Semen was collected from camels showing signs of rut period, and the reaction time, copulation time, and seminal parameters were evaluated. Data obtained was analysed by using the computer program SPSS (version 25; IBM Corp.), and means and standard errors were compared. Male reproductive behaviours (protrusion of soft palate, poll gland secretions, frequent urination, increased frothy salivation) were more pronounced in MLT-treated camels (83.33%) than in control camels (50%). Reaction time and copulation times were lower (112.81±30.06s and 229.8±37.91s) in MLT-treated camels than in controls (359.00±41.35s and 301.89±93.90s). Higher semen volume was observed in MLT-treated (5.34±2.06mL) than in control (3.11±1.75mL) camels (P

In seasonal breeders, reproductive seasonality can have a substantial influence on the efficiency of assisted reproductive technologies. This study assessed seasonal effects on cleavage and blastocyst rates, as well as on quality of invitro-produced (IVP) goat embryos over 18 months. In total, 2348 (autumn: 811, spring: 404, summer: 639, and, winter: 494) cumulus–oocyte complexes (COC) were recovered from slaughterhouse ovaries during 49 replicates (autumn: 17, spring: 7, summer: 15, and, winter: 10) and matured in TCM-199 with 10ng mL−1 epidermal growth factor and 100µM cysteamine for 22h at 38.8°C (5% CO2 in air). Matured oocytes were fertilized with frozen/thawed semen in synthetic oviductal fluid (SOF) with 10% oestrus sheep serum. Sperm and oocytes were co-incubated for 16h at 38.8°C in a humidified atmosphere of 5% CO2 in air. Presumptive zygotes were cultured in SOF medium supplemented with bovine serum albumin (3mg mL−1) for 8 days at 38.8°C in a humidified atmosphere of 5% O2, 5% CO2 and 90% N2. At Day 2, 10% fetal calf serum was added to the culture droplets. The embryos produced were fixed and stained with Hoechst to count their total number of cells, under an epifluorescence microscope. The results of cleavage and blastocyst rates, including hatching rate, from each routine of IVEP were considered as replicates. These data were tested for normality by the Shapiro-Wilk test, before being subjected to ANOVA, followed by Tukey HSD test. The odds ratio (OR) among seasons (autumn: breeding; spring: anoestrus) were calculated. Values of P 0.05) to all others. Indeed, greater possibility of cleavage was observed in autumn (OR: 2.43) and summer (OR: 2.39) compared with spring. The blastocyst rate from cleaved embryos was greater (P 0.05) in the embryo hatching (mean: 66±2.0%) and blastocyst cell number (mean: 193±2.0 cells). In conclusion, the breeding season (autumn) leads to improved oocyte developmental competence, resulting in greater cleavage and blastocyst yield, whereas embryo quality remained similar throughout the year. Further studies at the molecular level might indicate the mechanisms involved and provide clues to alleviate the negative effect of season.

The objective of the present study was to observe age-related differences in seminal parameters and expression of fertility marker genes in Marwari stallions. Semen was collected from 9 Marwari stallions (6 ejaculates per stallion) of 3 different age groups (2 to 4 years, GI; 4 to 6 years, GII; and >6 years, GIII) twice a week using an artificial vagina by allowing the stallions to mount a mare. The collected ejaculate was divided into 2 parts. Part A was used for evaluation of various seminal parameters (colour, consistency, total volume, gel volume, gel-free volume, pH, progressive sperm motility, sperm concentration, sperm viability, total sperm morphological abnormalities, plasma membrane integrity, acrosomal integrity, and DNA integrity). Part B was centrifuged to obtain the sperm pellet for DNA extraction. Six fertility-related marker genes (SPATA1, SP17, PLCz, CRISP3, UBB, and PRM1) were examined in the spermatozoa DNA using PCR. Expression levels of these genes were also studied using quantitative real-time PCR. Data obtained were analysed statistically by one-way or two-way analysis of variance using the SPSS computer program (version 20.0; IBM Corp.). No significant difference was observed among the GI, GII, and GIII stallions for seminal parameters, except mean sperm concentration, which was lowest in 2- to 4-year-old stallions (146.06±11.50 million mL−1), intermediate in stallions >6 years old (182.03±8.51 million mL−1), and highest in stallions of 4 to 6 years (270.92±9.12 million mL−1; P

The objective of this work was to evaluate pregnancy rates in buffaloes subjected to fixed-time AI (FTAI) during the year in Argentina. The data analysed was collected between 2016 to 2019 in a farm located in Paso Florentin City (27°20′33″ latitude and 58°08′27″ longitude) in Corrientes, Argentina. The animals were grazing native pastures (Andropogon lateralis, Paspalum almum). Ten fixed-time AI (FTAI) sessions were performed, 6 within the favourable season (BS, March–June) and 4 in the outbreeding season (OBS; September–December). Murrah and Mediterranean breed (n=141) buffaloes were used. All animals were healthy, without anatomical alterations and with normal reproductive tracts. Heifers (H), adult females with calf (AFC), and dry buffaloes (DB) were used. The average (±s.d.) weight of the animals were 459.0±37.5kg, 581.3±53.6kg, and 583.3±53.1kg, respectively. The presence of corpus luteum (CL) or a dominant follicle (≥8mm) at the beginning of the protocol was recorded. BS ovulation induction: Ovsynch (n=182); Day 0 gonadotrophin-releasing hormone (GnRH; 100μg, busereline acetate), Day 7 prostaglandin F2α (150μg, cloprostenol), Day 9 GnRH and FTAI 16h after the last GnRH injection. For the OBS (n=85): Day 0 intravaginal device (700mg of P4) + oestradiol benzoate (0.2g), Day 8 removal of the device + prostaglandin F2α + oestradiol cypionate (0.1g) + eCG (400IU) and Day 10 FTAI (52–56h) after device removal. Semen from 5 bulls of proven fertility was used, and the same technician performed all the inseminations. A specifically designed format to record the data were used and the average±s.d. of the physiological and the meteorological parameters were calculated. Pregnancy was detected 40 days after FTAI using ultrasonography. Comparison within variables were performed using ANOVA and Tukey test, with InfoStat-Statistical Software. A total of 267 inseminations were performed, and the overall pregnancy rate (PR) was 37.45% (100/267). Comparing BS with OBS, PR were 40.7% (74/182) and 30.6% (26/85) (P0.05]. The presence of a CL at the beginning of the FTAI protocol had a positive effect on PR during the year in all categories [31.79% (55/173) H, 40.42 (38/94) AFC, 21.51 (17/79) DB vs. 25.45% (28/110) H, 29.68 (19/64) AFC, 19.56 (9/46) DB; P

The results of AI depend on many factors, with the quality of semen being one of the most important. Not all male hybrids can meet the requirements for semen quality, because they often have reduced fertility following cryopreservation. Thus, it is necessary to improve semen processing before use in AI. The aim of the study was to evaluate the effectiveness of using the “swim-up” flotation method to improve sperm quality of hybrid males of the Ovis genus. Semen from interspecific hybrid rams (1/4 Argali×3/4 Romanov, n=15; 1/8 Argali×7/8 Romanov, n=15) was freshly obtained, frozen–thawed, and processed by the swim-up method. Evaluation of sperm motility was determined using computer-assisted semen analysis. Statistical analysis was performed using SPSS vs.15.0 (ANOVA and t-test; SPSS Inc.). Semen was collected during the breeding season (October–December) via artificial vagina. Assessment of acrosome integrity was determined using differential staining with a Diachem diff-quick kit (NPF ABRIS+). The degree of sperm DNA fragmentation was determined using the acridine-orange test. The sperm freezing/thawing cycle was accompanied by sperm damage and an increase in the proportion of immobile sperm from 10 to 58%, with non-progressive movement increasing from 9 to 19.3%. The number of spermatozoa with abnormal morphology doubled, and the DNA fragmentation index increased from 16 to 26%. Use of the swim-up procedure allowed us to sort progressively motile spermatozoa. The content of progressively motile spermatozoa in the samples obtained from the supernatant was 86%, which was 2.3 times higher than in frozen–thawed sperm (P≤0,01). The obtained results show the effective use of the swim-up procedure to determine the quality of semen in hybrid rams. These studies were carried out with financial support from the Russian Science Foundation, grant No. 18-16-00079 and the Ministry of Science and Higher Education of the Russian Federation.

Interspecific hybrids, especially males, are sterile or have reduced fertility. The structure and development of reproductive organs is species-specific. The study of development stages, morphology and biometric parameters in the male testes, the characterisation of species features, and the development of assisted reproductive technologies is of great importance. The aim of this research was to study morphometric parameters of the testes of interspecific hybrids of Argali with sheep of the Romanov breed compared with the original Romanov breed. The objects of research were purebred males of the Romanov breed (n = 8) and hybrid males with the following genotypes: 1/4 Argali × 3/4 Romanov (Group I, n = 10), 1/8 Argali × 7/8 Romanov (Group II, n = 12). The collection of testes from purebred and hybrid animals was carried out at castration (age = 12 months). Testicular tissue was fixed in Bouin’s solution. The following metrics were evaluated: weight and morphometric parameters of the testis, diameter of the seminiferous tubules, and the number of spermatogenic cells in the seminiferous tubule. Statistical analysis (comparison between groups with t-test and ANOVA) was performed using SPSS vs.15.0 (SPPS Inc.). The weight of testes in purebred Romanov males averaged 393 ± 41 g. In hybrid animals from groups I and II, this indicator was lower compared with that of purebred animals, by 58% and 41% (P

The present study aimed to evaluate an oestrous synchronization protocol and oestrus response before AI in cows from an organised communal production system. A total of 74 cows Bos indicus (Brahman) and Bos taurus/indicus hybrid (Nguni) type from different communal farmers were screened based on: age (3 to 8 years), body condition score of ≥3 (1–5 scale), not pregnant (excluding heifers), 90 days postpartum, number of parities, good mothering ability, and negative for contagious abortion. Selected cows were subjected to a 9-day OvSynch+CIDR protocol. In brief, the protocol included the insertion of controlled intravaginal drug release (CIDR®, Pfizer Laboratories) devices in the reproductive tract (vagina) containing 1.9g of progesterone in each cow on Day 0 with first oestradiol benzoate (Oestradiol benzoate®, VTech) 2-mL intramuscular (IM) injection. Pregnant mare serum gonadotrophin (PMSG; Chronogest®, Intervet International B.V.) 2.5-mL (IM) injection on Day 5. Removal of CIDR and (IM) injection of prostaglandin F2α (PGF2α; Estrumate®, Intervet South Africa (Pty.) Ltd.) 2mL on Day 8. Following CIDR removal, a heat mount detector (Kamar®) was applied on the individual cow’s tail head for oestrus observation (signs of heat) with second oestradiol benzoate 1-mL (IM) injection on Day 9. AI was performed 36h following withdrawal of the CIDR. Frozen/thawed semen from Bonsmara bulls (n=2) of known and proven fertility was used for AI. The GameteTek Cryo-Mobile laboratory was used during thawing of semen straws. and sperm motility and morphological traits were immediately evaluated by computer-assisted sperm analysis (Sperm Class Analyzer®) before each individual cow was inseminated. Pregnancy diagnosis was performed 90 days after timed AI by ultrasound and transrectal palpation. Data were analysed using the logistic regression procedure of SAS (SAS Institute Inc.), with synchronisation response and conception being treated as binary response variables. All cows were synchronized successfully and an oestrus response rate of 100% was recorded. Pregnancy rates were similar (37.8% vs. 38.9%) for both Bos indicus and Bos taurus/indicus hybrid cattle. Bulls total sperm motility of ≥75% were recorded, following thawing of semen straws. Thus, there was no effect of bull on pregnancy. In conclusion, acceptable oestrus synchronization response was achieved in this communal setup. Superior genetic materials can be successfully introduced through assisted reproductive technologies in organised communal production systems.

Invitro embryo production (IVP) is an important tool for genetic improvement in small ruminants. Semen quality is one of the most important aspects to consider for the success of this assisted reproductive technique. With ovine IVP, it is a common practice to use fresh semen for IVF, but this could be a problem because the differences between ejaculates from the same animal are well documented and a source of variation in IVP results. The objective of this research was to compare the effect of fresh and frozen–thawed domestic sheep (Ovis aries) semen on IVF for ovine IVP. The research was carried out in the reproduction laboratory at the Palominos Ranch (Jalisco, Mexico). The IVP was performed with a continuous invitro culture system. Ovaries (n = 186) were collected from a slaughterhouse (Leon, Mexico) and transported to the laboratory within 2 h in physiological saline solution (0.9% NaCl) supplemented with penicillin G (100 IU mL−1) and streptomycin sulphate (100 µg mL−1). For IVP, IVF-Bioscience™ media were used for IVM, IVF, and invitro culture (IVC). For IVM, the cumulus–oocyte complexes (COCs) were selected (only grades 1 and 2) and matured for 24 h at 38.5°C in 5% CO2 in air and 100% humidity. Matured oocytes (n = 1000) were invitro fertilized using either fresh or frozen–thawed semen (Triladyl™; Minitube) from the same sheep, at a concentration of 2 × 106 sperm mL−1, for 18 h in 38.5°C, 5% CO2 in air, and 100% humidity. The presumptive zygotes were denuded by pipetting and set in IVC until Day 7 at 38.5°C, 5% CO2, 5% O2, and 90% N2 at 100% humidity. The percentages of cleavage, embryos with more than 6 cells, and blastocysts on Day 7 of culture were evaluated, based on the initial number of oocytes entering into IVM. Statistical analyses were carried out with the GLM procedure of SAS software (version 9.3; SAS Institute Inc.) to evaluate the results of fresh versus frozen–thawed (α level = 0.05). Rates of cleavage, embryos with more than 6 cells, and blastocysts on Day 7 were similar (P > 0.05): fresh 52.3 ± 3.0%, 43.6 ± 2.6%, and 34.3 ± 2.9%, respectively; frozen–thawed: 53.3% ± 3.0, 41.1 ± 2.6%, and 33.3 ± 2.9%, respectively. In conclusion, under the conditions of this research, the use of fresh and frozen–thawed semen had similar results for ovine IVP.

Climate change represents a major stressful environmental condition that compromises the reproductive efficiency of animals. The temperature affects various components of the maturation processes in cattle oocytes, this includes disruptions of oocyte development and maturation. Therefore, the study aimed to compare the effects of different incubation temperatures (32.5, 33.5 34.5, 35.5, 36.5, 37.5, 38.5, 39.5, 40.5, 41.5, 42.5, and 43.5°C) on the maturation rate of cattle oocytes. Cattle ovaries were collected from a local abattoir and transported to the laboratory in thermo flask at 37°C. Oocytes were recovered from ovaries using the aspiration method and matured using 500 uL of TCM-199 medium supplemented with 10% fetal bovine serum, FSH, LH, and E2 covered with mineral oil. The maturation rate of oocytes was determined by the extrusion of the first polar body after 22h of IVM period. The total number of 80 oocytes were matured in each group and replicated 4 times. Data were analysed using GenStat® statistical program (VSN International). Comparisons were considered significantly different (P

Oestrus and ovulation synchronization (E/OS) regimens for fixed-timed breeding are useful when consistently eliciting ovulation. Early synchronization in the reproductive season promotes unreliable oestrus behavioural and physiologic response due to insufficient ovarian priming. In ruminants, equine (eCG) or human chorionic gonadotrophin (hCG) has FSH bioactivity or elicits ovulation, correspondingly. Hence, 120 and 60 IU of eCG and hCG, respectively, are included in goat E/OS regimes. This experiment addresses the time when eCG/hCG (CG) is given relative to progestagen (P4) withdrawal and its effect on oestrus and ovulation. Fourteen non-lactating, Alpine breed goats, ranging from 1 to 6 kiddings, averaging ± s.d. 3.9 ± 2.0 years of age, and 56.3 ± 5.0 kg of bodyweight (BW), and body condition score (BCS) of 2.6 ± 0.2, were evaluated early in the transitional reproductive phase (12 h daylight:12 h darkness). E/OS was accomplished with 12 day of P4 (200 mg) exposure by intravaginal insert. Three treatments were evaluated: Traditional (T; n = 4) did not receive CG and served as the control group; early (E; n = 5) received CG 24 h before P4 removal; and reverse (R; n = 5) received PGF2α 24 h before P4 withdrawal as well as CG concurrent with P4 withdrawal. Oestrus response to E/OS was evaluated 24 h after breeding using epididymectomized bucks. Ovaries were scanned by transrectal ultrasound (Aloka SSD-500V/7.5-MHz linear array probe) for 4 consecutive days starting with the first i.m. injection of 1.0 mL of PGF2α or 1.5 mL of CG. Images were digitized (MediCapture™) for later morphometry. The absence of effect (P > 0.05) from concomitant variables age, BW, BCS, and parity was ascertained using a logistic regression model (JMP/SAS v15; SAS Institute Inc.). Ovulation, defined as the disappearance of the largest (>7 mm) preovulatory follicle on a subsequent observation, was 100, 80, and 100%; and the average ± s.e. number of ovulations per goat was 2 ± 0.41, 1 ± 0.32, and 1.3 ± 0.49 (P > 0.05). Table 1 summarises follicular size documented of 122 observations of ovulatory areas. Graafian follicle location was not different for left or right ovaries (P > 0.05). Oestrus behavioural response to each E/OS treatment (T, E, or L) up to 24 h after P4 removal was 50, 80, and 80% (P > 0.05), respectively. In summary, oestrus response and ovulation were not affected by the variables studied. In this experience the timing of CG was not relevant. Table 1. Mean diameter (± SE; mm) of nonovulatory and preovulatory follicles on left (L) and right (R) ovaries E/OS protocol Non- and preovulatory Nonovulatory Preovulatory L R L R L R Traditional 6.4 ± 0.6 5.5 ± 0.6 4.8 ± 0.4 4.5 ± 0.4 7.7 ± 0.4 8.9 ± 0.3 Early 5.2 ± 0.3 5.5 ± 0.4 4.9 ± 0.2 4.4 ± 0.3 8.4 ± 1.2 8.1 ± 0.2 Reverse 6.6 ± 0.5 5.3 ± 0.6 5.1 ± 0.4 4.4 ± 0.5 8.7 ± 0.3 8.1 ± 0.2 Overall 5.7 ± 0.19 4.7 ± 0.14 8.3 ± 0.14

Reproductive performance is one of the key factors in livestock production. It is well known that reproductive traits are influenced by several genetic factors, such as the increase of individual inbreeding levels, which are associated with changes in sperm motility and shape in several species. In horses, the increase in inbreeding is a common problem because of the reduction in effective population size and the increase in selection intensity observed in several breeds. However, studies assessing the effect of high levels of inbreeding on the sperm quality of stallions are scarce. In the present study, we aimed to determine the effect of increased inbreeding levels and age on the sperm motility patterns of Purebred Spanish horses (PRE). We performed kinetic characterisation of 557 sperm samples of 82 PRE stallions aged between 3 and16 years, using computer-assisted sperm analysis (Androvision™, Minitube). We evaluated 5 parameters in 6 different fields per sample: curved line velocity (VCL, µm/s), velocity average path (VAP, µm/s), velocity straight line (VSL, µm/s), amplitude of lateral head displacement (ALH, µm), and beat-cross frequency (BCF, Hz). We determined the pedigree-based inbreeding coefficient (Fped) based on ∼300,000 PRE pedigree records to evaluate the inbreeding effect. Individuals were separated into 2 groups: highly inbred (n=339) and lowly inbred (n=218) according to an F value of 12.5%. Differences between groups were analysed using a generalized linear model. The analysis did not show significant differences (P>0.05) in the variables analysed with respect to the age of stallions. However, VAP, VCL, and AHL were lower in highly inbred than in lowly inbred animals (P0.05) were observed between the two study groups. In conclusion, age did not affect sperm quality parameters in the age group of stallions analysed. In addition, we demonstrated that high inbreeding coefficient reduced the mean velocity and trajectory pattern of spermatozoa in PRE.

Oocyte recovery is a reproductive technology that can be done by using two techniques, aspiration and slicing. Invitro maturation (IVM) is an additional reproductive technology used to advance an oocyte to a maturation stage; thereafter, it may be used during IVF. The objectives of the present study were (1) to compare two different oocyte retrieval methods (aspiration and slicing) from pig ovaries on oocyte quality and quantity, and (2) to compare three different IVM media [NCSU 37, TCM-199, and modified porcine follicular fluid (mpFF=porcine follicular fluid+FSH+LH] on oocytes’ polar body extrusion. During aspiration, an 18G needle was attached to a 10-mL syringe and all visible follicles were aspirated. During slicing, a surgical blade was used to slice the ovaries held in mDPBS. Follicular fluid collected from both methods was assessed for the presence of oocytes with the aid of a microscope. The collected oocytes were then categorized as Grade A, B, or C: Grade A=oocytes with compacted, multilayered cumulus cells and a homogeneous ooplasm; Grade B=oocytes with a compact cumulus cell layer with homogeneous ooplasm; Grade C=oocytes with a less compact cumulus cell layer with irregular ooplasm containing dark granules. The IVM media were placed in a four-well multidish; thereafter Grades A and B oocytes were allocated per treatment groups and matured for 44h. The treatment means were compared using the Fisher’s protected t-test least significant difference. The results showed significant differences between the grades of oocytes (P0.05). In conclusion, the slicing method proved to be better than aspiration with regards to the retrieval of Grades A and B oocytes as well as the total number of oocytes retrieved. The TCM-199 and mpFF media had a higher percentage of oocytes with polar body extrusion than NCSU 37.

Domestic cat invitro embryo production (IVEP) begins with IVM of oocytes to produce mature oocytes; that is, MII. The domestic cat (Felis catus) has been used as a model to carry out assisted reproductive technology (ART) research for application in wild feline species that may be threatened or endangered. The objective of this research was to evaluate oocyte maturation of domestic cats in different reproductive stages: (1) prepubertal, (2) oestrus, (3) pregnant, and (4) anoestrus. The present study was carried out at the Universidad Autónoma Metropolitana Unidad Xochimilco in Mexico City. Unless otherwise stated, all reagents used were from Sigma-Aldrich. The domestic cat ovaries were obtained from a veterinary clinic using salpingo-oophorectomy hysterectomy (OSH). Ovaries were classified as one of the following: (1) prepubertal (female cats under 6 months of age); (2) in oestrus (one or more 2-mm mature follicles); (3) pregnant (presence of fetuses with one or more corpora lutea; and (4) anoestric (ovaries without follicular activity). The ovaries were transported (0.05). This study shows that the domestic cat reproductive stage does not significantly affect the production of mature oocytes for use in ART.

The Dorcas gazelle (Gazella dorcas), in the family Bovidae, is one of the smallest species of the genus Gazella. Its population is decreasing and it is classified as vulnerable by the International Union of Conservation of Nature Red List of Threatened Species. The subspecies Saharan Dorcas gazelle (G. dorcas osiris), originating in the central-western Saharan and Sahel regions, is almost extinct in the wild because of over-hunting. Thereon, assisted reproductive techniques (ARTs) could help in its population management. Here, we performed invitro maturation (IVM) of oocytes recovered from an adult female of Saharan Dorcas gazelle (10 years old) to attempt IVF with frozen-thawed epididymal sperm cells from an adult male (9 years old) of the same subspecies, following protocols for ARTs in cattle. Epididymal sperm was obtained after postmortem examination by cannulation of vas deferens with commercial semen extender Gent A (Minitub). Motility features were assessed using a computer-assisted sperm analysis system (Proiser SL), and viability was evaluated using eosin/nigrosin staining. After collection, viability (88.3%), total (17.8%) and progressive (8.7%) motility were assessed. A total of 820.5×106 sperm cells was obtained and diluted (1:1) in a semen extender with permeable cryoprotectants (Gent B, Minitub). Then, the sample was refrigerated in straws (200×106 mL−1; 0.5mL) at 4°C for 1h and exposed to liquid nitrogen vapors (LN2) for 10min before being directly plunged into and kept on LN2. A total of 35 oocytes were recovered by slicing, 27 of which were selected for IVM (24h, 38.5°C, 5% CO2 in humidified air). Motile sperm were obtained by centrifugation of thawed semen (10min, 1000×g) by performing a density gradient (BoviPure, Nidacon). Post-thawing viability (72.9%) and total (14%) and progressive (4%) motility were evaluated just before IVF. After IVM, 17 oocytes were incubated for IVF for 20h with 1×106 thawed sperm cells mL−1 (although the first polar body extrusion was observed only in 5 of the 17 oocytes before IVF; 29.4%). Also, 7 oocytes selected for IVM were vitrified and kept for the genetic bank, and 3 were fixed. Thereafter, all oocytes that underwent IVF were incubated for invitro embryo culture (38.5°C, 5% CO2, 5% O2 in humidified air) to avoid discarding any potential zygote. Cleavage was assessed at 48h post-IVF. From those, only 2 oocytes successfully achieved IVF out of the 5 that showed first polar body extrusion (40%), although cleavage was asymmetric and embryos did not progress. The rest of oocytes (12) did not achieved IVM, IVF, or cleavage. Results suggest that optimal protocols and appropriate individualized conditions for vulnerable nondomestic ungulates are required for successful ARTs. Future efforts are needed to improve preservation strategies for the gametes/embryo conservation of endangered species. JG and MRC contributed equally to this work. JG is supported by AGAUR (2018 FI_B 00236) and MRC by FPU (Training for Academic Staff) (FPU15/06029).

Sperm selection methods are routinely applied to prepare semen for IVF in animal species. These procedures are used to improve sperm quality characteristics as well as to remove other background material and debris. Percoll gradient is widely used in animal IVF laboratories. Different Percoll gradient concentrations and volumes can be used to improve the sperm sample motility percentage. The objective of this research was to evaluate the effect of 2 different Percoll concentrations for ovine IVF sperm selection and effects, if any, on invitro embryo production (IVP). Specifically, Percoll gradients consisted of Group 1 (G1) 40–80% and Group 2 (G2) 45–90%, 400µL each. The research was carried out in the reproduction laboratory at Palominos Ranch (Jalisco, México). The IVP was performed with a continuous invitro culture system. Ovaries (n=157) were collected from a slaughterhouse (León, México) and transported to the laboratory within 2h in physiological saline solution (0.9% NaCl) supplemented with penicillin G (100IU mL−1) and streptomycin sulphate (100µg mL−1). For IVP, IVF Bioscience™ media were used for IVM, IVF, and invitro culture (IVC). For IVM, the cumulus–oocyte complexes (COCs) were selected (only grades 1 and 2) and matured for 24h at 38.5°C in 5% CO2 in air and 100% humidity. Matured oocytes (n=800, divided equally into 4 replicates) were divided into 2 groups, G1 and G2. The IVF process was conducted with semen selected through a mini-Percoll technique with gradients at a concentration of 45% (top layer) and 90% (bottom layer) or 40% (top layer) and 80% (bottom layer) for G1 and G2, respectively, using frozen–thawed semen from the same ram, at a concentration of 2×106 sperm mL−1, for 18h in 38.5°C, 5% CO2 in air, and 100% humidity. The presumptive zygotes were denuded by pipetting and set in IVC until Day 7 at 38.5°C, 5% CO2, 5% O2, and 90% N2 at 100% humidity. The percentages of cleavage, embryos with more than 6 cells, and blastocysts on Day 7 of culture were evaluated, based on the initial number of oocytes entering into IVM. The statistical analyses were carried out with the GLM procedure of SAS software (version 9.3; SAS Institute Inc.) to evaluate the results of G1 versus G2 (α level=0.05). Cleavage rate was 47.8%±2.5 and 55.9%±2.5, respectively, in G1 and G2. The percentages of embryos with more than 6 cells were 38.1%±2.2 and 43.5%±2.2, respectively, in G1 and G2. The percentage of blastocysts on Day 7 was 27.4%±1.1 and 37.3%±1.1, respectively, in G1 and G2. There were no significant differences between groups (P>0.05) for percentage of cleavage and embryos with more than 6 cells, although the percentage of cleavage tended to be greater for G2 (P=0.06). Additionally, G2 had a larger percentage of blastocysts on Day 7 compared with G1 (P

The recovery of an exceptionally high number of cumulus–oocyte complexes (COC) in a single ovum pickup (OPU) session is rare, and thus seldom reported in the literature. In the present report, we describe one of these cases. The present data refers to an OPU session performed by author A. O. Resende in a commercial beef farm, located in the rural area of the city of Barretos, Brazil, on October 15, 2019. Breed heifers (N=12) underwent OPU at a random stage of oestrous cycle without any hormonal treatment. Follicle aspirations were performed following the standard operation procedures (SOP) established by ABS Pecplan OPU team, using a portable ultrasound (DP2200, Mindray do Brasil) equipped with a 7.5-MHz microconvex probe, a vacuum pump (WTA), and 18 gauge disposable Jelco catheter (Smith Medical). Invitro embryo production (IVP) was performed at Invitro Technologies laboratory, ABS Pecplan Uberaba, located 140km from the farm. On this day, 1312 oocytes were recovered from the 12 donors. However, a single heifer produced 485 viable COC, from over 500 total COC recovered. The COC from this donor were distributed in four groups, which underwent IVF with Y-sorted semen from four Senepol sires. The invitro embryo production was performed following the SOP established by invitro Technologies, ABS Pecplan. Embryo production was compared among sires by the chi-squared test. Of the 4 sires used during IVF, sire number 4 resulted in a higher blastocyst rate compared with the others (sire 1=25.4%, sire 2=30.6%, sire 3=33.9%, and sire 4=46.3%; P

One of the parameters that has the most influence on invitro embryo production programs is the number of oocytes obtained in each follicular aspiration session (ovum pickup; OPU). A significant impact of the breed of the donor on this parameter has been reported. The objective of this work was to compare the parameters of an invitro embryo production program of cows of different breeds in Paraguay. A total of 4811 OPU sessions were performed between 2016 and 2019 in cows classified according to breed and type. Beef-type breeds: Nelore (Bos indicus): n = 1569; Aberdeen Angus (Bos taurus): n = 1276; and Brangus (Cross): n = 182 OPU, and Gyr (Bos indicus): n = 1086; dairy breeds: Holstein (Bos taurus): n = 401, and Girolando (Cross): n = 297. The oocytes were aspirated and transported to the same laboratory located in Asuncion, Paraguay. The invitro embryo production protocol was similar in all types. Bull semen tested for IVF was used. The number of oocytes, percentage viability, and percentage embryo production were evaluated. Additionally, the mean and standard deviation of the variables were calculated to show the efficiency of the processes, according to the production aptitude of the donors (meat or milk) and the breed type (Bos indicus, Bos taurus, or crosses). Continuous data were analysed with ANOVA and Tukey’s post hoc comparisons. Categorical data were analysed using Chi-squared at a significance level of 0.05. In this work, the production aptitude had no significant effect over the studied variables. However, some significant differences were found considering the breed type of the oocyte donor cow, as shown in Table 1. When crosses with the pure breeds are compared with their crosses, we found that Brangus produced more pregnancies and a greater number of oocytes/OPU compared with Angus (P

SexedULTRA 4M™ (Select Sires) is made using an improved method of sex-sorting sperm in a less damaging environment for retaining sperm integrity through the sorting process. The combination of invitro embryo production (IVP) and sexed semen technologies has been successful for intensity selection in the cattle industry. The objective of this research was to compare conventional (CONV) and SexedULTRA 4M (ULTRA-4M) semen for bovine IVP using 2 bulls. The research was carried out in the reproduction laboratory at Palominos Ranch (Jalisco, Mexico). The IVP was performed with a continuous invitro culture system. Ovaries from commercial cattle (n = 213) were collected from a slaughterhouse (Leon, Mexico) and transported to the laboratory within 2 h in physiological saline solution (0.9% NaCl) supplemented with penicillin G (100 IU mL−1) and streptomycin sulphate (100 µg mL−1). For IVP, IVF Bioscience™ media were used for IVM, IVF, and invitro culture (IVC). For the IVM, the cumulus–oocyte complexes (COCs) were selected (grades 1 and 2) and matured for 24 h at 38.5°C in 5% CO2 in air and 100% humidity. Matured oocytes (n = 1200, divided equally into 3 replicates) were divided into 2 groups, the CONV group and the ULTRA-4M group. The IVF process was conducted with both CONV and ULTRA-4M semen from the 2 bulls, separately, at an adjusted concentration of 2 × 106 and 0.5 × 106 sperm mL−1, respectively, for 18 h in 38.5°C, 5% CO2 in air, and 100% humidity. The presumptive zygotes were denuded by pipetting and set in IVC until Day 7 at 38.5°C, 5% CO2, 5% O2 and 90% N2 at 100% humidity. The cleavage rate, embryos with more than 6 cells, and blastocysts on Day 7 of culture were evaluated. Statistical analysis was carried out with the GLM procedure of SAS (version 9.3; SAS Institute Inc.) to evaluate the results of CONV versus ULTRA-4M for each bull (α level = 0.05). Cleavage rates were 57.1% ± 1.5 and 59.4% ± 1.5, respectively in CONV and ULTRA-4M groups with Bull 1 and 43.3% ± 1.5 and 45.2% ± 1.5 with Bull 2. The percentages of embryos with more than 6 cells were 51.4% ± 1.0 and 53.9% ± 1.0, respectively, with CONV and ULTRA-4M with Bull 1 and 30.4% ± 1.0 and 33.4% ± 1.0 with Bull 2. The percentage of blastocysts on Day 7 with Bull 1 was 34.4% ± 1.7 for CONV and 36.2% ± 1.7 for ULTRA-4M; for Bull 2, the results were 26.5% ± 1.7 for CONV and 29.5% ± 1.7 for ULTRA-4M. There were no significant differences between the CONV and ULTRA-4M groups (P > 0.05) for all variables analysed for each bull; however, Bull 1 was significantly superior to Bull 2 for all variables analysed. In conclusion, under the conditions of this research, ULTRA-4M and CONV semen produced similar bovine IVP results.

The first commercial production of sexed semen was at the Cogent company in the United Kingdom. Since then millions of offspring have been born using sexed semen produced by flow cytometry. Sexed semen technology has recently been modernized to what is now known as ULTRA (ST Genetics), completely modifying the technique, medium, and sperm concentration. In field trials using AI, there was no difference between conventional semen (CONV) and ultra-sexed semen at a concentration of 4 million sperm per straw (ULTRA-4M). The combination of in vitro embryo production (IVP) and sexed semen technologies has been successful for intensity selection in the cattle industry. The objective of this work was to compare the CONV and ULTRA-4M semen for bovine IVP. The research was carried out in the reproduction laboratory at the Facultad de Estudios Superiores Cuautitlan-Universidad Nacional Autonoma de Mexico (FESC-UNAM). The IVP was performed with a continuous in vitro culture (IVC) system. Ovaries (n = 213) were collected from a slaughterhouse (Queretaro, Mexico) and transported to the laboratory within 2 h (FESC-UNAM) in physiological saline solution (0.9% NaCl) supplemented with penicillin G (100 IU mL−1) and streptomycin sulfate (100 µg mL−1). For IVP, VITROGEN media were used for IVM, IVF, and IVC. For the IVM, the cumulus-oocyte complexes were selected (only grades 1 and 2) and matured for 24 h at 38.5°C in 5% CO2, 95% air, and 100% humidity. Matured oocytes (n = 1000) were divided into two groups, the CONV group and the ULTRA-4M group. The IVF process was developed with CONV and ULTRA-4M semen from the same bull (Holstein) at a concentration of 2 × 106 and 0.5 × 106 sperm mL−1, respectively, for 18 h in 38.5°C, 5% CO2, 95% air, and 100% humidity. The presumptive zygotes were denuded by pipetting and left in IVC until Day 7 at 38.5°C, 5% CO2, 5% O2, and 90% N2 at 100% humidity. The cleavage rate, embryos of more than 6 cells, and blastocysts on Day 7 of culture were evaluated. The statistical analysis was carried out with the GLM procedure of the SAS software (version 9.3; SAS Institute Inc.) to evaluate the results of CONV vs. ULTRA-4M (α level = 0.05). Percent cleavage for CONV was 72.2% ± 2.53 and 75.6% ± 2.53 for ULTRA-4M. For embryos with more than 6 cells, the results for CONV and ULTRA-4M were 59.8% ± 5.61 and 62.8% ± 5.61, respectively. The percentage of blastocysts on Day 7 was 37.8% ± 5.39 for CONV and 43.6% ± 5.39 for ULTRA-4M. There were no significant differences between the groups (P > 0.05) for all variables analysed. Although the number of blastocysts on Day 7 were numerically higher in the ULTRA-4M, differences were not significant. In conclusion, under the conditions of this research the ULTRA-4M had similar results as CONV for bovine IVP.

The alpaca (Vicugna pacos) is a ruminant livestock species in the South American camelid family. There are more than 9 million South American camelids globally that make important contributions to the livelihoods of rural farmers through conversion of low quality roughages to high quality food and fibre. Reproductive biotechnologies for alpacas are not well developed compared with those for other ruminant livestock species. In particular, embryo cryopreservation technologies are lacking. The objective of this study was to evaluate under field conditions a vitrification protocol originally developed for old world camels that we adapted for use in alpacas. Potential donors were evaluated for follicular development using a 7.5-MHz ultrasound probe. Hembras (sexually mature female alpacas) with ovarian follicles 7-10 mm in diameter were behaviour tested to determine sexual receptivity, and receptive females were naturally mated to a proven herd sire. At the time of breeding, non-superovulated donors (n = 4) received 30 μg gonadorelin. Embryos were nonsurgically collected 7 days after breeding and handled at 20°C. Diameter of harvested embryos (n = 4 quality grade 1 hatched expanded blastocysts) was measured using an eyepiece reticle. All recovered embryos were placed individually into 0.5-mL drops of vitrification solution (VS1: 1.4 M glycerol) for 5 min, 0.5-mL drops of VS2 (1.4 M glycerol + 3.6 M ethylene glycol) for 5 min, 0.05-mL drops of VS3 (3.4 M glycerol + 4.6 M ethylene glycol) for 20 s, and 0.05-mL drops of VS3 for 20 s while loading into open-pulled straws (OPS). Each OPS was plunged directly into liquid nitrogen for storage for 29 days. At warming, each OPS was submerged into a 1-mL drop of warming solution 1 (WS1: 0.5 M galactose) for 1 min followed by 1 min in WS2 (0.25 M galactose) for 5 min before being incubated at 37°C in 5% CO2 in humidified air for 21 h in 1 mL of Syngro holding medium supplemented with 10% (vol/vol) alpaca serum. Embryos that grew during culture (n = 2) were transferred individually into synchronous recipients, and embryos that did not appear to grow (n = 2) were transferred together as a pair. Prior to embryo transfer, potential recipients were evaluated ultrasonographically as described previously. Hembras with ovarian follicles 7-10 mm in diameter were behaviour tested, and sexually receptive females received 30 μg gonadorelin 6 days before embryo transfer. Final selection of recipients (n = 3) was based on presence of a corpus luteum and nonreceptive behaviour to a herd sire 24 h before transfer. Pregnancy was detected ultrasonographically, and fetal heartbeat was detected 29 days post-transfer in one of the three recipients. Ultrasound at 177 days post-transfer revealed that the pregnancy, generated from a 400 μm × 375 μm vitrified-warmed embryo that had grown in culture, was still ongoing. If this pregnancy results in the birth of a live cria (newborn alpaca), it would represent-to the best of our knowledge-the world's first cria born from a cryopreserved alpaca pre-implantation embryo. It would also demonstrate the potential utility of this protocol under field conditions.

The menstruating spiny mouse is the first rodent identified to exhibit natural spontaneous decidualisation, cyclical endometrial shedding and regeneration. While the spiny mouse shares several primate-like characteristics in its reproductive biology, it has not been established whether pseudopregnancy can be induced or if its cycles can be synchronised as in non-human mammals. Here we describe attempts to induce pseudopregnancy and synchronisation of menstrual cycles (i.e. Whitten effect) in spiny mice. Virgin females (n=3–8 per group) underwent one of the following procedures to induce pseudopregnancy: daily vaginal lavage only (control), progesterone injection, mechanical stimulation of the cervix and sterile mating. A separate cohort was also exposed to male-soiled bedding to assess the Whitten effect. Pseudopregnancy was deemed successful if females presented with extended (>12 consecutive days) leukocytic vaginal cytology. No female from any method of induction met this criterion. In addition, the menstrual cycles of a group of six females could not be synchronised, nor immediate ovulation induced via exposure to male-soiled bedding. These responses indicate that the spiny mouse does not behave as a typical rodent. Like higher-order primates, the spiny mouse exhibits a relatively rare reproductive strategy, of failure to show pseudopregnancy or cyclical synchronisation. This is further endorsement of the use of this species as a versatile animal model for translational studies of menstruation and fertility.

This report describes a recent meeting of the Endocrine and Reproductive Biology Society of Western Australia (ERBSWA) on 23 July 2019, and summarises the research presented and the prizes awarded.

Under natural conditions, mammalian oocytes and zygotes are never exposed to daylight or artificial light. During assisted reproductive procedures such as invitro production (IVP), oocytes and pre-implantation embryos are exposed to light from time to time before being transferred to recipients. The detrimental effect of visible light is not directly related to the intensity and exposure time, but is also a function of the spectral composition of the light. Recently, a green pass light filter was used during all steps of bovine IVP. Results indicated the protective effect of the filter against harmful blue and infrared regions of the light (Korhonen et al. 2009 Hum Reprod. 24, 308-314; https://doi.org/10.1093/humrep/den432). Data regarding either blue or infrared light are missing. Therefore, the effect of environmental light will be examined on the developmental capacity of bovine oocytes and pre-implantation embryos produced invitro. Three different experimental conditions were set during all IVP steps: (i) light from the microscope filtered with a red pass filter, no artificial light, and no daylight, (ii) artificial light without day light, light from the microscope without a filter, and (iii) artificial and daylight, light from the microscope without a filter, as normally used in the laboratory (control). Bovine cumulus-oocyte complexes were recovered from abattoir-derived ovaries by the slicing method. After 24h of maturation, fertilisation was realised and after 19h of co-culture of cumulus-oocyte complexes and sperm, presumptive zygotes were cultured in SOFaa for 8 days. Cleavage and developmental rates were recorded at Day 3 and Day 7/8 (Day 0=IVF, 10 IVP runs). Blastocysts from all groups were individually stored at −80°C until analyses. Then RT-qPCR (at least 12 replicates) was performed as described previously (Stinshoff et al. 2014 Reprod. Fertil. Dev. 26, 502-510; https://doi.org/10.1071/RD12372) for gene transcripts related to cellular stress (BAX, BCL2L1, HSPA1A, SOD1) and embryo developmental capacity (SLC2A3). The morphological quality of expanded blastocyst was assessed via a differential staining procedure (with propidium iodide and Hoechst, 4 replicates) to determine the number of inner cell masses (ICM), trophectoderms (TE), and total cells and to calculate the ICM/TE ratio. Data were analysed by analysis of variance followed by a Tukey test. Cleavage and developmental rates were similar in all groups of embryos (P>0.05). The relative abundance of all analysed gene transcripts was significantly increased (P ≤ 0.05) in blastocysts stemming from settings (i) and (ii) compared with those from the control group. Blastocysts generated using only artificial light or artificial and daylight did show a significantly increased number of ICM and total cells (86.3±3.2, 56.3±1.4; 183.6±4.0, 161.8±2.8) compared with embryos produced under filtered microscope light without any other light (35.3±0.8; 133.8±1.4; P ≤ 0.05). The number of TE cells was similar (P>0.05) in blastocysts of all groups (i: 98.5±1.3; ii: 97.3±2.6; iii (control): 105.5±2.4). These data suggest that day light exposure might influence bovine embryos at the molecular level, whereas the morphological quality seems to be unaffected when compared with embryos exposed to only artificial light.

High ambient temperature induces an increase in bovine body temperature above the physiological homeothermic point, leading to impaired reproductive performance. Belgian Blue bulls are more susceptible to heat stress compared with most other cattle breeds. Therefore, the aim of this study was to investigate whether high ambient temperature affected bull semen quality and subsequent embryo development. For this purpose, semen samples were collected from six Belgian Blue bulls during August (14-28 days after three consecutive warm days with temperature-humidity index between 63.8 and 83.7) and March 2016 (temperature-humidity index between 35.9 and 47.4). After semen collection, volume, sperm concentration, and motility of fresh semen were assessed. Furthermore, frozen sperm samples were used to assess the motion parameters and morphological abnormalities using computer-assisted sperm analysis, viability and reactive oxygen species (hydrogen peroxide and superoxide) production using flow cytometry, and acrosome integrity and chromatin condensation using fluorescence microscopy. Afterward, blastocysts were produced (r=4) by conventional invitro methods for assessment of embryo development (Wydooghe et al. 2014 Reprod. Fertil. Dev. 26, 717-724; https://doi.org/10.1071/RD13043). Cleavage rate was determined 48h after fertilisation, and the blastocyst rates were determined on Days 7 and 8 postinsemination. Moreover, Day 8 blastocysts underwent differential staining in order to determine the numbers of the inner cell mass, trophectoderm, total cell number, and apoptotic cells ratio. The data set was analysed using the GLM procedure of SAS (SAS Institute Inc.). Normal distribution was checked using the UNIVARIATE procedure, and the Shapiro-Wilk test and arcsine square root transformation were used when required. Furthermore, Duncan's test was applied to determine the significant differences (P=0.05). In fresh semen, samples from the non-heat-stressed (NHS) group showed a higher sperm concentration compared with samples from the heat-stressed (HS) group (P=0.05), whereas semen volume and motility were not affected by heat stress (P>0.05). In frozen-thawed semen, total and progressive motility and straight-line velocity were lower in the HS group compared with the NHS group (P=0.05), whereas the generation of H2O2, percentages of aberrant chromatin condensation, total morphological abnormality, spermatozoa with bent tails, and distal protoplasmic droplets were higher in the HS group compared with the NHS group (P=0.05). The cleavage rate and blastocyst rates on Days 7 and 8 were higher (P=0.05) in the NHS group (70.4±1.13, 25.4±1.84, and 40.3±1.15, respectively) compared with the HS group (62.8±1.49, 15.4±1.56, and 23.3±1.84, respectively). However, there were no significant differences in hatched or hatching rate of two treatments (P>0.05). The total cell number and trophectoderm were higher in NHS-derived blastocysts than in HS-derived blastocysts (P=0.05), whereas the apoptotic cells ratio was lower (P

The Canadian dairy industry has been using invivo and invitro assisted reproductive technologies to produce embryos. Technological improvements have helped increase the number and quality of embryos produced, but genetic and genomic tools for improving these traits have yet to be assessed for the Canadian Holstein population. Genetic parameters and a genome-wide association study were performed in Canadian Holstein for the total number of embryos (NE) and the number of viable embryos (VE). Results showed potential for genetic selection for both NE and VE, with heritability estimates (± s.e.) of approximately 0.15±0.01. Genetic correlations between the number of embryos produced using different procedures (invivo and invitro) suggested that a similar number of embryos should be expected from a donor regardless of the procedure used. A region on chromosome 11 of the bovine genome was found to be significantly associated with the number of embryos, indicating a potential regulatory role of this region on embryo production. Overall, these findings are of interest for the Canadian dairy industry because they provide useful information for breeders that are interested in producing embryos from the elite donors in their herds or in the population using assisted reproductive technologies.

Reproductive traits in domestic pigs are one of the most important economic characteristics for improved pig production. Seminal plasma (SP) constituents provide a better indicator of male fertility as measured by semen traits. The objective of this study was to compare spermatozoa traits, testosterone concentration, SP constituents, and lipid peroxidation in Large White×Landrace (LW×LR) and Kolbroek boars aged between 8 and 9 months. Kolbroek (n=12) and LW×LR (n=12) boars were used in this study. Semen was collected using the gloved-hand technique. Blood samples were collected from each boar monthly to determine testosterone concentrations. Spermatozoa motility traits was evaluated using computer-aided sperm analysis. Spermatozoa viability was evaluated using the hyperosmotic swelling test, lipid peroxidation was evaluated using the malondialdehyde (MDA) test, and SP protein was evaluated using atomic absorption spectrophotometric analysis. Analysis of variance was used to test the difference between the breeds. The LW×LR boars had higher ejaculation volume and testosterone concentrations (P0.05). The average genotype spermatozoa total motility was 91.7±4.0 and 90.6±4.1%, respectively (P>0.05). No significant differences were found between genotype in abnormal spermatozoa morphology, rapid spermatozoa, and total spermatozoa motility, spermatozoa velocity on the curve line, and hyperosmotic swelling test. Spermatozoa with distal, head, and midpiece abnormalities were significantly different in Kolbroek boars (17.1±1.4, 5.0±2.0, and 7.0±2.6%, respectively) and LW×LR boars (9.2±0.1, 3.5±1.1, and 2.6±1.2%, respectively). The LW×LR boars had lower levels of MDA than Kolbroek boars (P

Alpacas as other camelids are induced ovulators and require an external stimulus-mounting by the male-for ovulation. NGF-β, protein present in the seminal plasma (SP) is reported as being responsible for stimulating ovulation. However, limited information exists on the effect of ovulation inducers in the new follicular wave emergence with application on protocols of superstimulation. The aim of the study was to determine the effect of two ovulation induced techniques and two different times on the interval to application of equine chorionic gonadotrophin (eCG) for the ovarian superstimulation for the recovery of COCs by ovum pick- up (OPU). Alpacas were evaluated by transrectal ultrasonography with an Aloka SSD 500 ultrasound and transducer (7.5 MHz) to determine the presence of a dominant follicle = 7 mm. A 2 × 2 experimental design was used with adult female alpacas, 6 to 8 years old, which were assigned to one of the treatments when a dominant follicle = 7 mm was present. Alpacas were assigned randomly 1 of 2 groups (Day 0) in which follicular ovulation was induced by seminal plasma 1 mL IM (SP, n = 13; group 1) with application of 650 IU of eCG at 36 h (n = 6) or 48 h (n = 7) post-application of SP, or by GnRH with 0.008 mg of Buserelin IM (GnRH, n = 10; group 2) with application of 650 IU eCG at 36 h (n = 5) or 48 h (n = 5) post-application of GnRH. On Day 7, COCs were counted and the OPU procedure was performed in every group. Data were analysed using ANOVA. Results are presented in Table 1. In conclusion, both inducers of ovulation and both times of application of eCG were effective for producing follicles of 7 mm and COCs acceptable for recovery in Alpacas. Table 1.Number of follicles = 7 mm (top) and number of COCs recovered (mean ± s.e.m.) Outcome and group 36 h 48 h Total Follicles GnRH 6.8 ± 1.93 13.4 ± 4.34 10.10 ± 2.50a SP 5.5 ± 2.23 5.29 ± 1.15 5.38 ± 1.25b Total 6.09 ± 1.44 8.67 ± 2.18 7.43 ± 1.33 COCs GnRH 5 ± 0.97 3 ± 1.26 4.33 ± 0.74a SP 1 ± 0.41 0.33 ± 0.22 0.67 ± 0.25b Total 3.29 ± 0.67x 1.4 ± 0.6y 2.5 ± 0.53 a,bValues within columns with different letters differ significantly (P = 0.05). x,yValues within columns with different letters differ significantly (P = 0.05). This research funded by CIENCIACTIVA-CONCYTEC as part of the project title “Role of seminal plasma in reproductive physiology and application of biotechnologies in camelids” (149-2017).

Compared with conventional semen, use of sexed semen decreases the conception rate (CR); therefore, it is important to address the factors that lead to sperm damage. Recently, a novel sexed semen technology was developed for improving dairy cattle fertility (Betthauser et al. 2016 J. Dairy Sci. 99 (E-Suppl. 1), 534). However, there are few reports about the fertility of this novel sexed semen in dairy cattle in Japan. The objective of this study was to compare the CR of traditional sorting semen (S1: flow cytometry/cell sorting) and the novel sexed semen (S2: SexcelTM, ABS Global Inc.) in Holstein heifers and cows in Japan. The CR for the first insemination was obtained from 391 Holstein cows and 148 heifers (10.7-17.8 months old) from 14 dairy farms in Hokkaido from June 2017 to April 2019. Semen used for AI was collected from six bulls and packaged into 0.25-mL straws and frozen. Cows inseminated with conventional frozen semen (S3: unsexed semen in 0.5-mL straws) were used as controls. Calving data were collected from 123 cows and 69 heifers that had calved between January 2018 and May 2019. The diagnosis of pregnancy was confirmed using ultrasonography between 30 and 45 days after insemination. The CR and sex ratio were analysed using chi-square test. The average parity number was 1.9±1.1, and average days open was 84.9±20.4 days. The average interval between calving and the first service was 86.2±20.3 days. The average milk yield (at 305 days) was 12 195±1595 kg. All of the animals were inseminated after the onset of standing heat or removal of the tail chalk of natural heats and oestrus and ovulation synchronisation programmes on the farm. Conception rates of heifers of the first service of S1 (n=54) and S2 (n=94) were 68.5 and 70.2%, respectively. Conception rates of cows of the first service of S1 (n=32), S2 (n=173), and S3 (n=186) were 56.3, 57.8, and 53.8%, respectively. There were no significant differences in CR between these groups (P>0.4). The proportion of female calves produced using S1 (n=61) and S2 (n=57) was 91.8 and 93.0%, respectively, which was significantly more (P0.8) in terms of the proportion of calves produced. The results of this study demonstrated that the traditional sorting and novel sexed semen (S1 and S2, respectively) did not differ in terms of the first-service CR and produced considerably more female calves than when the conventional semen (S3) was used. Furthermore, these findings indicate that the use of this novel sexed semen could achieve a sex ratio close to 90% female offspring.

The development of markers for reproductive function has been a goal for the embryo production industry and reproductive biology as a method to improve success and increase the ability to explain reproductive phenomena. Cattle and buffalo are bovines that show different reproductive parameters raised in the same conditions. The aim of this work was to compare the results of the invitro production of cows (Bos indicus) and buffalo (Bubalus bubalis) from animals kept under the same conditions of feeding and handling. This study was conducted in an Argentinian commercial herd of buffalo and cattle located in the province of Corrientes (−27.742859 latitude, −57.773611 longitude) during the breeding season of 2018 (March-May). Twenty animals of each species were used. Anti-Mullerian hormone (AMH) levels of each animal were determined using ELISA. Standardized protocols were used for oocyte aspiration, maturation, fertilisation, and culture of the embryos, and frozen semen of a single proven bull was used in each species. The numbers of follicles, oocytes, and embryos were recorded and analysed individually and grouped by species (Table 1). The normality of the data was evaluated with the D’Agostino and Shapiro-Wilk tests and the comparisons between species using the Mann-Whitney and ANOVA tests, and correlation analysis was performed using the Spearman test. Values are shown as median and range. A P-value 0.05). There were no significant differences in the number of follicles and the quality of the oocytes. Significant differences were found in the number of oocytes cleaved of 4 (0-17) vs. 0.5 (0-4) and blastocysts per animal of 1.5 (0-15) vs. 0.1 (0-2) for cows and buffalo, respectively. The number of blastocysts in relation to the number of oocytes cleaved did not show statistical significance. In spite of the difference in the AMH levels and the embryo production parameters, which were lower in buffalo, no correlation was found between AMH and the parameters studied in both species. The results obtained here are confirmed by others in the literature. Levels of AMH as a marker for embryo production are not useful in cattle and buffalo and do not explain the differences observed, certainly AMH is a marker for quantity as the preserver of the ovarian reserve. Table 1.Comparison of individual values of AMH levels, oocyte quality, and embryo production between cattle and buffalo Parameter Median (range) P-value Cattle Buffalo AMH (pgmL−1) 688.5 (45.3-2394) 73.8 (14.8-262.5)

Current methods of mammalian semen evaluation focus on determining spermatozoa motility, concentration, mitochondrial status, and nucleus or chromatin structure integrity, quantifying their ability to bind to ova or measuring seminal plasma content of various biochemical markers. However, there is a paucity of studies that address relationships between sperm head morphometry (the external shape and dimensions of the sperm) and fertilising ability. Sperm head morphometrics are influenced by many molecular and biochemical factors such as genetics, DNA or protein condensation, and cell membrane permeability, all of which can affect semen viability. The objective of this experimental work was to determine quantitative correlations between sperm head dimensions and various indices of sperm quality in frozen-thawed ram semen. Ejaculates were collected from 16 clinically healthy rams (4 Polish Lowland (PON), 4 Olkuska, 5 synthetic line BCP (Berrichon du Cher×Charolais×PON/Polish Merino), and 3 synthetic line SCP (Suffolk×Charolais×PON/Polish Merino) aged 4-12 years) into an artificial vagina in the middle portion of the breeding season. Ejaculates from each ram were divided into two equal portions, diluted with a commercial semen extender prepared in deionised water or nanowater (water declusterised using cold plasma treatment) to a final concentration of 400×106 spermatozoamL−1, and frozen in 0.25-mL plastic straws. After 6 months of being cryogenically preserved, semen samples were thawed and used for the preparation of smears stained with eosin or SpermBlue. Images of the samples containing at least 100 spermatozoa were taken under 200× magnification and used for determination of sperm head morphology with the image analytical software Image Pro Plus (Media Cybernetics Inc.). Sperm progressive motility and survival time, as well as extender concentrations of alkaline phosphatase and aspartate aminotransferase, were measured. Finally, 128 BCP ewes were inseminated laparoscopically with the ram semen and fertility parameters were recorded. The present data were analysed using a multivariate analysis of variance in SAS (SAS Institute Inc.) and Spearman correlation tests. There were no significant effects or interactions of breed, staining method, or extender diluent on sperm head dimensions (head length, width, area, perimeter, and roundness). The mean head length was negatively correlated (P

Bapedi is a fat-tailed, hardy, and disease-tolerant breed of sheep that is indigenous to South Africa. The Bapedi sheep breed is facing genetic degradation due to nonselective cross-breeding and irregular mating; therefore, breeding strategies for urgent conservation of Bapedi sheep are crucial. The aim of the present study was to investigate the relationship between body measurements and semen traits of Bapedi rams conserved in situ (conserved in their natural habitat) and ex situ invivo (live animals conserved outside their natural habitat). Semen was collected twice per week from 33 rams aged 2-4 years (four ejaculates per ram) using an electroejaculator. Body measurements (rump height, body length, scrotum circumference (SC), scrotal width, and scrotum length (cm)), body condition score (BCS; scale of 1-5), bodyweight (BW; kg), rectal temperature (°C), semen volume (mL), spermatozoa concentration (billionsmL−1), and motility parameters were measured using a computer-aided semen analysis system. Data were analysed using analysis of variance and the general linear models of SAS (SAS Institute Inc.). The Pearson correlation coefficient in SAS was used to determine the relationship among variables. There was uniformity in all body measurements of Bapedi sheep regardless of the conservation method (P>0.05). There were no significant differences in body temperature during semen collection, SC, semen volume, semen pH, semen concentration, spermatozoa total motility, and kinematics in Bapedi rams between methods of conservation (P>0.05). Pearson correlation revealed moderate significant relationships between semen volume and BW, BCS, and SC (r=0.315, 0.638, and 0.381, respectively). Rump length had a moderate correlation (0.566) with normal spermatozoa (P

The 2019 meeting of the Society for Reproductive Biology (SRB) provided a platform for the dissemination of new knowledge and innovations to improve reproductive health in humans, enhance animal breeding efficiency and understand the effect of the environment on reproductive processes. The effects of environment and lifestyle on fertility and animal behaviour are emerging as the most important modern issues facing reproductive health. Here, we summarise key highlights from recent work on endocrine-disrupting chemicals and diet- and lifestyle-induced metabolic changes and how these factors affect reproduction. This is particularly important to discuss in the context of potential effects on the reproductive potential that may be imparted to future generations of humans and animals. In addition to key summaries of new work in the male and female reproductive tract and on the health of the placenta, for the first time the SRB meeting included a workshop on endometriosis. This was an important opportunity for researchers, healthcare professionals and patient advocates to unite and provide critical updates on efforts to reduce the effect of this chronic disease and to improve the welfare of the women it affects. These new findings and directions are captured in this review.

In vitro embryo production (IVP) can increase the reproductive potential and genetic quality of cattle, as well as other species. This powerful assisted reproduction tool can be used to produce embryos from prepubertal calves, reducing the generation interval. A recent sexed semen technology known as ULTRA (ST Genetics), completely modified the technique, the media and sperm concentration. In field trials with AI there was no difference between conventional semen (CONV) and ultra-sexed semen at a concentration of 4 million per straw (ULTRA-4M). The combined use of IVP and ULTRA-4M can decrease the selection time for improving dairy and beef cattle herd genetics. The objective of this research was to compare the CONV and ULTRA-4M semen using bovine IVP and prepubertal calves. The research was carried out in the reproduction laboratory of the Facultad de Estudios Superiores Cuautitlan - Universidad Nacional Autonoma de Mexico (FESC-UNAM). The IVP was performed with a continuous in vitro culture (IVC) system. The ovaries were collected in Campeche, Mexico, from Bos indicus × Bos taurus crossbred calves (6 months old) using surgical castration (for export to the United States) and transported to the laboratory (FESC-UNAM) in BO-HEPES-IVM (Bioscience™), in an oocyte transporter (WTA). Vitrogen media were used for IVF and IVC. For IVM, the cumulus-oocyte complexes (COCs) were selected (only grades 1 and 2) and matured for 24 h at 38.5°C. Matured oocytes (n = 600, divided equally into five replicates) were divided into 2 groups, the CONV group and the ULTRA-4M group. The IVF process was conducted with CONV and ULTRA-4M semen from the same bull (Holstein) at a concentration of 2 × 106 and 0.5 × 106 spermatozoa mL−1, respectively, for 18 h in 38.5°C, 5% CO2, 95% air, and 100% humidity. The presumptive zygotes were denuded by pipetting and set in IVC until Day 7 at 38.5°C, 5% CO2, 5% O2, and 90% N2 at 100% humidity. The cleavage results were recorded 56 h after the beginning of IVC. The cleavage rate, embryos with more than 6 cells, and blastocysts on Day 7 of culture were evaluated. The statistical analysis was carried out with the GLM procedure of the SAS software (version 9.3; SAS Institute Inc.) to evaluate the results of CONV vs. ULTRA-4M (α level = 0.05). The percentage of cleavage for CONV was 46% ± 1.4 and 43.2% ± 1.4 for ULTRA-4M. The results for embryos with more than 6 cells in the CONV and ULTRA-4M groups were 16% ± 0.6 and 14% ± 0.6, respectively. The percentage of blastocysts on Day 7 for CONV was 9% ± 0.6 and 8% ± 0.6 for ULTRA-4M. There were no significant differences between groups (P > 0.05) for all variables analysed. In conclusion, under the conditions of this research the ULTRA-4M and CONV produced similar results for IVP.

The objective of this study was to evaluate the correlation between the number of corpora lutea (CL), as determined by B-mode ultrasonography, and the total number of structures recovery by non-surgical embryo recovery (NSER) from superovulated ewes. Somali (n=18), Santa Inês (n=18), and Morada Nova (n=16) ewes received an intravaginal device with progesterone (0.33g, controlled internal drug release device, CIDR, Zoetis), which was maintained for 9 days. The superovulatory protocol started 60h before device removal, with 6 decreasing doses (25, 25, 15, 15, 10, and 10%) of p-FSH (IM 133mg, Folltropin V, Vetoquinol) injected every 12h. D-Cloprostenol (IM 37.5μg, Prolise, Agener Union) was injected 12h before and at the moment of device removal. Ewes in oestrus were mated by fertile male rams three times after the onset of oestrus with a 12-h interval. Flunixin meglumine (24.9 mg; Banamine, MSD Animal Health) was injected IM on Days 12, 13, and 15. One day before NSER, B-mode ultrasound evaluations were performed using portable equipment (Z5 Vet, Mindray) with a stiffened multifrequency linear probe to evaluate the number of structures present in the ovaries. Embryo recovery was performed 7 days after progesterone CIDR removal (Day 16). The number of CL and the number of recovered structures were determined and their association evaluated using the Pearson correlation test (P

An experiment was designed to evaluate pregnancy rates per AI (P/AI) in suckled beef cows synchronised with a progesterone (P4)/oestradiol-based protocol and AI with non-sexed (conventional) semen and with a sexed-sorted semen with 65% X-bearing sperm and 35% Y-bearing sperm that was named SuperConventionalTM. Angus and Hereford suckled cows (n=558), 60-90 days postpartum, with a body condition score of 2 to 3.5 (1-to-5 scale) and 48% (269/558) with a corpus luteum, were randomly allocated to be AI with non-sexed (conventional) or SuperConventional semen. The experiment was performed on 3 farms using straws with sexed-sorted SuperConventional semen containing 6 or 8 million sperm and non-sorted conventional semen with 25 million sperm per straw. Semen was from split ejaculates from two Angus bulls. All cows received a P4 device with 0.6g of P4 (Pluselar, Calier) and 2mg of oestradiol benzoate (Calier) on Day 0. All P4 devices were removed on Day 8, and all cows received 400IU of equine chorionic gonadotrophin (Vetegon, Calier), 150µg of d+cloprostenol (Veteglan, Calier), and 1mg of oestradiol cipionate (Calier) at the same time. All cows were also tail-painted and observed for signs of oestrus. Cows with >30% of the tail paint rubbed off by 48-50h after P4 device removal were AI at that time with either SuperConventional or conventional semen. Those not showing oestrus by 48-50h received 10µg of buserelin (Pluserelina, Calier) at that time and were AI with either SuperConventional or conventional semen 50-52h after P4 device removal. Pregnancy was diagnosed using ultrasonography 60 days after AI, and all pregnancies were sexed to determine the proportion of female pregnancies. Data were analysed using GLM for binary data. The P/AI were different between cows showing or not showing oestrus (P

This study examined the effect of genotype and extender diluent on the characteristics of frozen-thawed ram semen. Twenty ejaculates collected from 10 rams aged 4-12 years (2 Polish Lowland (PON), 4 synthetic line BCP (Berrichon du Cher×Charolais×PON/Polish Merino), and 4 SCP (Suffolk×Charolais×PON/Polish Merino) in mid-breeding season were divided into six equal portions each and frozen in semen extender prepared by mixing commercial Triladyl extender (MiniTub GmbH) with deionized water (Aqua Purificata, Prolab) or nanowater (NW; deionized water declusterized in a cold plasma generator for 15, 30, 45, 60, or 90 minutes (15’, 30’, 45’, 60’, or 90’, respectively); Nanotechnology Systems) and Gallus domesticus egg yolk (1:3:1 vol/vol/vol). All semen samples were evaluated for progressive motility (Sperm Class Analyzer) and sperm morphology (Nikon Eclipse 80i microscope, Nikon Corp.). In addition, the ex situ survival time at 37°C and extender content of ALT (alanine transferase), ALP (alkaline phosphatase), and aspartate aminotransferase were measured, and the numbers of apoptotic, necrotic, and live spermatozoa were determined post-thawing with flow cytometry (BD Accuri™ C6 Plus, Becton Dickinson). Data were analysed by two-way analysis of variance and least significant difference test (SigmaPlot, Systat Software Inc.). The PON exceeded (P

Pigs are the only livestock species encoding a functional protein for both the second isoform of gonadotrophin-releasing hormone (GnRH-II) and its cognate receptor (GnRHR-II). Unlike the classical GnRH system (GnRH-I and GnRHR-I), GnRH-II and GnRHR-II are abundantly produced in porcine testes. Moreover, GnRH-II binding its receptor on Leydig cells stimulates luteinizing hormone-independent testosterone secretion. Interestingly, GnRHR-II is also localised to the connecting piece of mature, ejaculated spermatozoa, whereas GnRH-II is detected in seminal plasma, an interaction possibly influencing the function of sperm. To examine the role of GnRH-II and its receptor in the testis, we produced a swine line with reduced endogenous GnRHR-II levels (GnRHR-II KD). The objectives of this study were to (1) compare sperm characteristics between mature GnRHR-II KD and littermate control boars on the day of collection and following semen extension and (2) determine whether a GnRHR-I and GnRHR-II antagonist alters sperm characteristics after storage of extended semen. In Experiment 1, GnRHR-II KD (n=3) and littermate control (n=3) ejaculates were collected (Day 1) and computer-assisted sperm analysis (CASA) was performed (IVOS II Animal; Hamilton Thorne) to determine measures of sperm motion (motility, progressive motility, slow, and static), morphology (normal morphology, bent tail, coiled tail, distal droplet, proximal droplet (PD), distal midpiece reflex, elongation, and area), and kinematics (length of average path (DAP), length of straight line path (DSL), length of curvilinear path (DCL), average path velocity (VAP), straight line velocity (VSL), curvilinear velocity (VCL), straightness (STR), linearity (LIN), amplitude of lateral head displacement (ALH), beat-cross frequency, and wobble (WOB)). Next, 3 billion sperm were extended with Androstar Plus (80-mL doses; Minitube) and stored at 17°C until Day 7 CASA. Data were analysed with the MIXED procedure of SAS (SAS Institute Inc.). On Day 1, semen doses from GnRHR-II KD boars had reduced DSL, VSL, STR, LIN, and WOB (P

Intensive reproductive management in dairy herds is mostly based on AI using high-merit bulls. Therefore, semen quality of bulls is of high importance. An association between semen quality and fatty acid content in feed has been suggested. Accordingly, the aim of this study was to examine the effect of omega-3 supplementation on sperm traits and fertilization competence. Fifteen Israeli Holstein bulls were assigned to three experimental groups. Bulls were fed over 13 weeks with a standard ration top-dressed with encapsulated-fat supplementation-fish oil (FO) or flaxseed oil (FLX; i.e. omega-3 sources), or saturated fatty acids (SFA, control). Ejaculates were collected before initiation of the study, during the feeding trial, and 1 month after feeding trial. Ejaculates were treated according to the routine procedure of the Israeli AI centre (Sion Ltd.), frozen, and stored in straws. Frozen-thawed samples were subjected to “swim-up” procedure, and spermatozoon viability, mitochondrial membrane potential, reactive oxygen species (ROS) level, acrosome membrane integrity, and DNA fragmentation were evaluated via flow cytometry, using sperm-specific kits (EasyCyte, IMV Technologies). Feeding with FO, FLX, or SFA did not affect the viability or mitochondrial membrane potential of sperm collected before, during, or after the feeding trial. On the other hand, a reduced proportion of sperm with ROS expression was recorded in the FLX samples compared to the SFA sample at the end of the feeding trial (42.2±1.2 vs. 47.3±4.3%, respectively; P

Mycoplasma bovis (Mbo) has been isolated from genital tracts of bulls, and it can survive in processed semen. Experimental studies have shown that Mbo inoculation into the uterus or insemination with Mbo-infected semen can cause bursitis, salpingitis, abortion, and infertility. The control of Mbo is very difficult because of latent carrier animals, increasing resistance to antibiotics, and unavailability of effective vaccines. The aim of this study was to follow the passage of Mbo infection from naturally contaminated semen to transferable embryos during bovine in vitro embryo production (IVP). (Unless otherwise stated, all chemicals used were purchased from Sigma-Aldrich.) Two batches of slaughterhouse-derived oocytes were matured in tissue culture medium 199 (TCM-199) with glutamax-I (Gibco™; Invitrogen Corporation) supplemented with 0.25 mM sodium pyruvate, 100 IU mL−1 penicillin, 100 µg mL−1 streptomycin, 2 ng mL−1 FSH (Puregon, Organon), 1 µg mL−1 β-oestradiol (E-2257), and 10% heat-inactivated fetal bovine serum (FBS; Gibco™) for 24 h at 38.5°C in maximal humidity in 5% CO2 in air. Matured oocytes were fertilized for 20 h in IVF-TL medium supplemented with 10 µg mL−1 of heparin and 2 mM of PHE at 38.5°C in maximal humidity in 5% CO2 in air, using spermatozoa per mL as a final concentration. The batches of oocytes were divided between uninfected IVP bull (N = 205) and naturally Mbo-infected AI bull (N = 690). Zygotes were cultured in G1/G2 media (Vitrolife) supplemented with bovine serum albumin, fatty acid free (4 mg mL−1), at 38.5°C in maximal humidity in 5% O2, 5% CO2, and 90% N2. Blastocysts were collected for Mbo cultures on Days 7 and 8 (IVF = Day 0). Samples of washed semen, fertilization medium, cumulus cells, culture medium, all wash media, and transferable embryos (with and without zona pellucidae) were collected for Mbo cultures. Half of the embryos were treated with trypsin according to IETS standards after the collections. The Mbo cultures were performed in accordance with procedures previously described by Bolske (1988 Zentralbl. Bakteriol. Mikrobiol. Hyg. A 69, 331-340), followed by detection with real-time PCR. Infection with Mbo does not seem to have negative effects on fertilization (cleavage rates: 77.1% and 89.0% for IVP and Mbo AI bulls, respectively) or embryo development rates (blastocyst rate: 26.3% and 32.5% for IVP and Mbo AI bulls, respectively). Following Mbo cultures, only washed semen was found to be Mbo positive via real-time PCR. We conclude that M. bovis is not likely transmitted in bovine IVP when using naturally infected semen. We acknowledge Tiina Kortelainen for technical assistance and the Ministry of Agriculture and Forestry for funding.

The objective of this study was to investigate a method of oviducal semen deposition as a strategy for producing offspring from poor-quality cryopreserved goat sperm. Invitro fertilisation (IVF) and AI are common assisted reproductive technologies used in small ruminants, but they have varied results in the goat. The use of poor-quality cryopreserved-thawed sperm (

Assisted reproductive technologies (ART) are being extensively used to produce cattle offspring. However, as shown by Siqueira et al. (2017 J. Dairy Sci. 100, 5899-5908; https://doi.org/10.3168/jds.2016-12539), phenotypical and performance differences between cows derived from distinct ART can be found at different stages of development. Thus, in an attempt to mimic the natural environment, reproductive fluids (oviductal (SOF) and uterine fluids) were added as supplementation to embryo culture media. Our hypothesis was that this improved culture media would produce calves more similar to the ones produced by AI. Invitro-produced (IVP) beef embryos were produced using SOF media supplemented with reproductive fluids (RF) or standard protocol (BSA), vitrified and later warmed and transferred to synchronized dairy recipients. Simultaneously, other dairy recipients were inseminated (AI) with the same bull used to produce IVP embryos. A total of 19 calves are included in this study (RF n=5, BSA n=7, AI n=7). Calves that did not reach 45 days of life were excluded from these data. All animals received the same feeding and housing conditions. Calves were examined at Days 0, 3, 7, 15, 30, and 45 of life. Each examination included weight, height at withers, thorax circumference, heart and respiratory rates, body temperature, and a blood sample from the jugular vein to perform a general haematological analysis (Siemens ADVIA 120) and glucose levels. A non-parametric test (Mann-Whitney U) was used to compare paired samples, with significance assumed when P

MRL/MpJ mice exhibit distinct phenotypes in several biological processes, including wound healing. Herein we report two unique phenotypes in the female reproductive system of MRL/MpJ mice that affect ovulation and luteinisation. We found that superovulation treatment resulted in the production of significantly more oocytes in MRL/MpJ than C57BL/6 mice (71.0±13.4 vs 26.8±2.8 respectively). However, no exon mutations were detected in genes coding for female reproductive hormones or their receptors in MRL/MpJ mice. In addition, the fertilisation rate was lower for ovulated oocytes from MRL/MpJ than C57BL/6 mice, with most of the fertilised oocytes showing abnormal morphology, characterised by deformation and cytolysis. Histological tracing of luteinisation showed that MRL/MpJ mice formed corpora lutea within 36h after ovulation, whereas C57BL/6 mice were still at the corpora haemorrhagica formation stage after 36h. The balance between the expression of matrix metalloproteinases and their tissue inhibitors shifted towards the former earlier after ovulation in MRL/MpJ than C57BL/6 mice. This result indicates a possible link between accelerated extracellular matrix remodelling in the ovulated or ruptured follicles and luteinisation in MRL/MpJ mice. Together, these findings reveal novel phenotypes in MRL/MpJ mice that provide novel insights into reproductive biology.

Reproductive biotechnology has continued to evolve rapidly, allowing the development of techniques to increase reproductive efficiency and contribute to the genetic improvement of cattle. Some of these techniques include the in vitro maturation and IVF of oocytes, sperm sexing and cloning. These modern assisted reproductive techniques can help produce offspring of desired genetic characteristics and of a pre-determined sex. However, studies of the bull’s contribution to in vitro reproductive performance are scarce in the Brahman breed. The aim of this study was to compare oocyte maturation and embryo production in vitro using frozen semen from 5 Brahman bulls (Bos indicus), cloned (n=1) and non-cloned (n=4), with characteristics and genetics of high commercial value. The age of the bulls at the time of semen collection and cryopreservation ranged from 2 to 7 years. The oocytes were obtained on 2 different dates (45 days between collections) using pooled oocytes collected by ovum pickup at random stages of the oestrous cycle, from a total of 15 Brahman donor cows. Oocytes were transported to a laboratory in the State of Chiapas, Mexico (Genemex Internacional). The oocytes were cultured in maturation medium for 24h. For IVF, conventional semen was used from one bull (B1) and his clone (B12), the grandson of B1 (B2), and 2 non-related bulls (B3 and B4). The gametes were co-incubated for 22h and afterward placed in medium for embryo development and cultured for 7 days in a humid atmosphere with 5% CO2 in air. Of the matured oocytes, 36/43 (84%), 16/32 (50%), 101/143 (70%), 46/67 (68%) and 53/65 (81%) were fertilized using semen from B1, B12, B2, B3 and B4, respectively. Of the fertilized oocytes, 15/30 (50%), 8/16 (50%), 45/101 (44%), 21/46 (45%) and 18/53 (34%) resulted in transferrable embryos, corresponding to semen from the same bulls, respectively. This would appear to be the first scientific report in Mexico about the use of semen from a cloned bull for in vitro embryo production. In IVF, similar results were observed between B1 and a non-related bull (B4). Similar results in transferrable embryos were observed between B1 and B12 but also similar to a related bull (B3) and a non-related bull (B4). A Fisher’s exact test of the IVF results comparing B1 and B12 found a significantly (P0.05) concerning the number of transferrable embryos produced by these two bulls. In conclusion, the Brahman bulls in this study differ in their contribution to IVF and embryo production. Further studies are required to determine the factors responsible for such effects, e.g. age differences or clone versus non-clone mosaicism. Results from this research contribute to the study and development of assisted reproductive techniques for increasing in vitro production efficiency in Zebu cattle. We thank the Rosales family, from El Herradero Ranch, in the State of Campeche, Mexico, for allowing the use of their cattle for this project.

Assisted reproductive techniques (ART) are widely used in domestic species, with increasing applications in wildlife conservation. However, the currently available techniques for semen preparations are not fully reliable or applicable in all species to ensure successful ART outcomes. Recent developments in nanotechnology offer new horizons for further optimization of sperm preparations. The use of conjugated magnetic nanoparticles allows for selective targeting and removal of moribund spermatozoa through the technique known as nanopurification, which has shown to be beneficial in domestic boars and bulls. Nanopurification is a rapid, straightforward, effortless, and noninvasive technique that, when applied in wildlife or endangered species, is expected to have a tremendous impact on ART outcomes. In this study, we evaluated the effectiveness of magnetic (iron oxide) nanoparticles designed to target moribund spermatozoa (e.g. acrosome reacted) on domestic felid and threatened bovid species. Fresh epididymal spermatozoa of domestic cats and ejaculated spermatozoa from genetically valuable threatened bulls (Ankole and Sahiwal) were collected and mixed with iron-oxide magnetic nanoparticles (MNP). After 30min of incubation at 38°C, semen mixtures were placed on an electromagnetic field (10-15min) to trap MNP-bound spermatozoa (moribund), followed by the elution of viable nontrapped spermatozoa (nanopurified). All samples were analysed for motility characteristics using a computer-assisted sperm analyzer. Aliquots of cat spermatozoa were subjected to (1) morphology and acrosome analyses (n=4-8 replicates), (2) cryotolerance to liquid nitrogen storage (n=4 replicates), and (3) IVF (n=1 replicate). Data were analysed with Student’s t-test and P0.05). Sperm MNP-bound samples exhibited higher proportions of moribund spermatozoa as compared to their nanopurified and control nonpurified counterparts (57.5v. 47.5 and 49.5%, respectively; P