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The addition of antioxidants to cryopreservation media reportedly improves sperm post‐thaw quality and reproductive performance after artificial insemination. Therefore, the objectives of this study were to evaluate if the addition of L‐carnitine and pyruvate to freezing media, or their addition to samples after thawing, improves the post‐thaw quality of equine spermatozoa. Thus, in Experiment 1, stallion semen samples were cryopreserved in: (1) EDTA–glucose–based extender with 20% egg yolk and 5% dimethylformamide (EDTA control); (2) skim milk–based extender with 20% egg yolk and 5% dimethylformamide (milk control); (3) Extender 1 supplemented with 50 mM L‐carnitine and 10 mM pyruvate (EDTA‐carnitine‐pyruvate); and (4) Extender 2 supplemented with 50 mM L‐carnitine and 10 mM pyruvate (milk‐carnitine‐pyruvate). In Experiment 2, 50 mM L‐carnitine and 10 mM pyruvate were added post‐thaw to samples cryopreserved with extenders 1 and 2 (EDTA control and milk control). Sperm kinematic parameters, DNA fragmentation, membrane lipid peroxidation, acrosome status and viability were evaluated after thawing. No significant differences (p > 0.05) were observed for most of the kinematic parameters, DNA fragmentation, membrane lipid peroxidation, acrosome status and viability of spermatozoa, between the samples frozen in the presence or absence of L‐carnitine and pyruvate, nor between the samples after the post‐thaw addition of these components. A higher (p < 0.05) mean velocity and higher (p < 0.05) amplitude of lateral head displacement were observed in the samples frozen in the milk‐based extender with the addition of L‐carnitine and pyruvate after thawing. The addition of 50 mM L‐carnitine and 10 mM pyruvate, either to the freezing extenders or after thawing, was not deleterious for sperm; however, it did not improve equine sperm motility, viability, acrosome and DNA integrity, nor decrease membrane lipid peroxidation after thawing. [ABSTRACT FROM AUTHOR]

This study aims to compare the efficacy of computer‐assisted sperm analysis (CASA) and smartphone‐applied sperm analysis (SASA) in assessing the quality of frozen–thawed bull semen. A total of 75 straws (n = 75) semen samples were used from different production batches of five Holstein bulls. The semen analyses were conducted in three groups: Group I (CASA‐37°C), semen samples were evaluated using the CASA system at 37°C (n = 25); Group II (SASA‐25°C), semen samples were assessed using the SASA system at a temperature of room heat (25°C) (n = 25); and Group III (SASA‐37°C), semen samples were evaluated using the SASA system at 37°C (n = 25). The frozen–thawed bull semen samples were analysed in terms of total motility (TM), progressive motility (PM), immotile, velocity average path (VAP), velocity curve linear (VCL), velocity straight line (VSL) and sperm concentration. There was no significant difference between the groups in terms of spermatozoa concentration (p >.05). However, significant differences among the groups were observed for total motile spermatozoa values (p <.001). Values of progressive motile spermatozoa were lower in Group I and Group II compared to Group III (p <.001). The immotile spermatozoa values were significant between the groups (p <.001) and were found to be proportional to total motile spermatozoa values. Additionally, the VAP, VCL and VSL values were comparable between Group II and Group III, but lower when compared to Group I. In conclusion, the results of the study demonstrate that the Sperm Cell™ system can accurately analyse the concentration of frozen–thawed bull semen. The analyses performed at room temperature indicate a parallelism between the PM value and CASA results. However, it is thought that SASA devices require a series of standardization studies in different semen extenders, different sample concentrations and different animal species, analogous to the standardization evolution process of CASA devices in semen analysis. [ABSTRACT FROM AUTHOR]

In vitro capacitation allows for a greater understanding of the mechanisms underlying fertilization and the development of improved reproductive techniques for improving fertility rates in porcine. Tyrodes albumin lactate pyruvate (TALP) and modified Krebs Ringers Broth (m‐KRB) are two medias that are commonly used in research experiments to induce capacitation in boar spermatozoa (Cañón‐Beltrán et al., Theriogenology, 198, 2023 and 231; Oberlender et al., Archivos de Medicina Veterinaria, 44, 2012 and 201; Sahoo et al., International Journal of Biological Macromolecules, 241, 2023 and 124502). Moreover, understanding the morphological and functional changes in boar spermatozoa at different hours of capacitation periods might aid in the development of novel techniques for improving sperm quality and increasing the litter size. This study was carried out to investigate the effect of Tyrode albumin lactate pyruvate and modified Krebs Ringers Broth media on in vitro capacitation of HD‐K75 boar spermatozoa at three different periods of incubation. A total of 24 ejaculate from four clinically healthy, 10–12 months aged HD‐K75 boars, maintained at ICAR‐All India Coordinated Research Project (AICRP) on pig were selected. Semen was collected by 'Simple fist' method using a portable dummy. The semen samples having 200 mL volume, 103 × 106 spermatozoa/ml concentration and 70% initial motility were selected and split into two parts and suspended in TALP and m‐KRB media, respectively, and incubated for 5 h at 37°C. Seminal parameters viz. sperm viability, plasma membrane integrity and acrosomal integrity were estimated in the samples at 0, 3 and 5 h of incubation. This study revealed that there was significant variation between media in live acrosome‐reacted (p <.05) and HOST‐reacted (p <.01) spermatozoa, while between capacitation periods significant (p <.01) variation was observed in hyperactivated spermatozoa, live acrosome‐reacted spermatozoa, HOST‐reacted spermatozoa, FITC‐labelled PSA, extracellular protein and sperm cholesterol. Non‐significant variation was observed in total phospholipid. TALP showed overall better consequence on sperm viability, plasma membrane and acrosomal integrity of boar spermatozoa. From this study, it could be concluded that both TALP and m‐KRB media were virtuous to induce capacitation in HD‐K75 boar spermatozoa. TALP media, however, had a better effect on sperm viability, plasma membrane and acrosomal integrity of boar spermatozoa. Out of the three different periods, 3 h capacitation period resulted in significantly (p <.01) higher incidence of sperm viability, plasma membrane and acrosomal integrity in HD‐K75 boar spermatozoa. [ABSTRACT FROM AUTHOR]

The Bachaur is a mediumized draft purpose breed which has been recognized by ICAR‐National Bureau of Animal Genetic Resources (NBAGR) Karnal, India, and presently is on the verge of extinction. Since there are no data regarding the seminal parameters of this breed, this work was performed to evaluate seminal parameters of freshly ejaculated semen. A total of three healthy breeding Bachaur bulls aged 2.5–5 years were selected for the study which were maintained under identical managemental conditions. Semen parameters of these bulls were studied across 10 ejaculates. The average scrotal circumference and testicular weight of the three bulls were 27.78 ± 1.2 cm and 400.67 ± 26.6 g, respectively. The average overall volume (mL), pH, concentration (million/mL), liveability (%), abnormality (%), HOST (%) and acrosome integrity (%) were 2.20 ± 0.19, 6.86 ± 0.06, 1245.60 ± 23.49, 85.09 ± 0.91, 4.13 ± 0.06, 81.16 ± 1.18 and 83.54 ± 1.32, respectively. The average overall mass motility of three Bachaur bulls was 3.57 ± 0.06 in 0–5 scale and individual motility averaged 84.78 ± 1.70 per cent. The volume of ejaculates in Bachaur bull seemed to be lower as compared to other exotic and Indian breeds. However, the semen parameters with regard to mass motility, liveability, abnormalities, hypo‐osmotic swelling test (HOST) and acrosomal integrity seemed similar to other exotic and Indian breeds. [ABSTRACT FROM AUTHOR]

The scenario of the fertile spermatozoa with high fertilizing capability is basically dependent on gene expression‐based epididymal function. The current investigation aimed to declare the varied expression of different candidate genes (PLA2G4D, LCN15, CLUAP1, SPP1, AQP12B, DEFB110 and ESR1) relevant to spermatozoa features between the different epididymal segments in the mature dromedary camels (n = 30). Scrotal contents were collected post‐slaughtering, during the breeding season and the epididymis was separated from the testicles and divided into three segments (caput, corpus and cauda) based on its morphology and anatomical characteristics. Epididymal spermatozoa were harvested from each epididymal portion and evaluated for motility, count, viability and morphology. Samples were grouped depending on their epididymal sperm cells features into high‐fertile (n = 15) and low‐fertile (n = 15) groups. The gene expression of the candidate genes was defined in the isolated RNA from each epididymal portion tissue. The segmental sperm motion and count were significantly (p <.05 and p <.01) higher in the three epididymal parts of high‐fertile camels than the lower ones. There were some candidate genes markedly up‐regulated in its expression in epididymal head of high‐fertile camels (PLA2G4D and LCN15) and low fertile (CLUAP1), while others in the body region of the high‐fertile group (SPP1, AQP12B and DEFB110). Nevertheless, ER1 did not differ in the expression among the epididymal segments. In conclusion, the variant expression patterns of these epididymal genes in relation to the regional spermatozoa features might suggest important roles of these genes in sperm maturation process in the epididymis and focusing more interest on their potential utility as markers for male camel fertility prediction. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the protective effects of nanoparticle selenium (SeNP) and sodium selenite (SS) on preventing oxidative stress during the freezing process of dog semen. A total of six dogs were used in the study. The ejaculate was collected from dogs three times at different times by massage method. A total of 18 ejaculates were used and each ejaculate was divided in five experimental groups. The experimental groups were designed to tris extender containing no antioxidants control, 1 μg/mL SeNP1, 2 μg/mL SeNP2, and 1 μg/mL SS1 and 2 μg/mL SS2. Extended semen were equilibrated for 1 h at 4°C, then frozen in liquid nitrogen vapour and stored in liquid nitrogen (~−196°C). After thawing, semen samples were evaluated in terms of CASA motility and kinematic parameters, spermatozoa plasma membrane integrity and viability (HE Test), spermatozoa morphology (SpermBlue) and DNA fragmentation (GoldCyto). Antioxidant enzyme activity (glutathione peroxidase; GPX, superoxide dismutase; SOD, catalase; CAT) and lipid peroxidation (malondialdehyde; MDA) were evaluated in frozen–thawed dog sperm. When the results were evaluated statistically, the progressive motility, VCL, and VAP kinematic parameters in the SeNP1 group were significantly higher than the control group after thawing (p <.05). The highest ratio of plasma membrane integrity and viable spermatozoa was observed in the SeNP1 group, but there was no statistical difference found between the groups (p >.05). Although the ratio of total morphological abnormality was observed to be lower in all groups to which different selenium forms were added, compared to the control group, no statistical difference was found. Spermatozoa tail abnormality was significantly lower in the SeNP1 group than in the control and SS2 group (p <.05). The lowest ratio of fragmented DNA was observed in the SeNP1 group, but there was no statistical difference was found between the groups (p >.05). Although there was no statistical difference between the groups in the evaluation of sperm antioxidant profile, the highest GPX, SOD and CAT values and the lowest lipid peroxidation values were obtained in the SeNP1 group. As a result, it was determined that 1 μg/mL dose of SeNP added to the tris‐based extender in dog semen was beneficial on spermatological parameters, especially sperm kinematic properties and sperm morphology, and therefore nanoparticle selenium, a nanotechnology product, made a significant contribution to the freezing of dog semen. [ABSTRACT FROM AUTHOR]

Artificial insemination (AI) centres select bulls as calves according to their genetic breeding values and raise them until the first semen collection; yet, a high dropout rate of reared bulls is a problem for AI centres. Potential hormonal indicators of bull sexual maturation (cortisol, dehydroepiandrosterone (DHEA), testosterone, oestradiol, insulin‐like growth factor 1 (IGF‐1)) were observed and evaluated in relation to the performance parameters to perhaps identify candidate biomarkers allowing an early selection of bulls as suitable sires. Blood samples from 102 German Holstein calves at 4 ± 1, 8 ± 1 and 12 ± 2 months of age from six AI centres were analysed using validated immunoassays for cortisol, DHEA, testosterone, oestradiol and IGF‐1. Semen analyses included native and thawed diluted semen. Bulls were classified at the first semen collection into groups with good versus poor performance (GP vs. LP). After 2 years, the subsequent differentiation was done in high (HPP), medium (MPP) and low performance persistency (LPP). Age at first semen collection was an important factor for sperm quality. Cortisol concentrations decreased with age, but the cortisol/DHEA ratio decreased with age only in GP bulls (p <.05). Oestradiol and testosterone concentrations both correlated with libido behaviour (p <.05). Testosterone and IGF‐1 concentrations were higher at the time of first semen collection in GP bulls and increased with age (p <.05). In conclusion, testosterone and IGF‐1 concentrations at first semen collection are associated with performance at first semen collection and future performance persistency, and might be useful early biomarkers for consistent sperm producing bulls on AI centres. [ABSTRACT FROM AUTHOR]

Dimensions of linear type traits facilitate selection of livestock for breeding and rearing. To date, use of linear type traits for selection of breeding bulls is highly concentric to scrotal circumference (SC), with probable overlook to other important traits. Present study reported the importance of various gonadal linear type traits on spermatozoa production, age‐related changes in gonadal linear type traits of bulls and predictive ability of these traits on bulls' reproductive potentials. Among all gonadal traits, testicular density (TD), scrotal volume (SV), paired testicular weight (PWT) and SC were found most important predictor variables in order, which can discriminate between good/poor breeding bulls, that is, produced frozen semen doses (FSD) or not. Dimensions of gonadal traits increased significantly up to 36 months age and thereafter, development became slow and negligible. In contrast, TD decreased by 30%, 51%, 64%, 68% and 71% at 12, 24, 36, 48 and >49 months age, respectively, from its base value at 6 months. Bulls of lower TD (≤0.88 g/cm3) had significantly higher ejaculate volume (+9%), sperm motility, sperm concentration (+100 million/mL) and sperm output (+26%)/ejaculate as compared to bulls of higher TD (>0.88 g/cm3). Discriminant function was developed using TD, SV, PWT and SC to identify bulls of superior reproductive potentials. It was concluded that among the investigated traits, TD was the strongest to discriminate between FSD and Non‐FSD bulls. Therefore, our findings suggested that TD could be more potential trait than SC for dairy bulls' breeding soundness evaluation and assessment of reproductive ability. [ABSTRACT FROM AUTHOR]

A variety of parameters, including liquefaction and semen viscosity, affect the sperm's ability to travel and reach the egg for fertilization and conception. Given that the details behind the viscosity of the semen in male camels have not yet been fully clarified, the purpose of this study was to ascertain how the addition of papain affected the viscosity of fresh diluted camel semen. The study examined semen samples derived from camels that had distinct viscosities. Sperm motility, viability, abnormal sperm percentage, concentration, viscosity, morphometry, acrosome integrity and liquefaction were among the evaluations following 0, 5, 10, 20 or 30 min of incubation at 37°C with papain (0.004 mg/mL, 0.04 mg/mL or 0.4 mg/mL; a semen sample without papain was used as a control). A statistically significant interaction between the effects of papain concentrations and incubation time was found (F = 41.68, p =.0001). Papain concentrations (p =.0001) and incubation times (p =.0001) both had a statistically significant impact on viscosity, according to a simple main effects analysis. A lower viscosity was found (p <.05) at 0.04 mg/mL (0.1 ± 0.0) after 10 min of incubation. A simple main effects analysis showed that papain concentrations and incubation time have a statistically significant effect on sperm motility (p =.0001). At 0.04 mg/mL papain, the sperm motility % was higher (p <.05) after 10 min (64.4 ± 4.8), 20 min (68.4 ± 6.2), and 30 min incubation (72.2 ± 6.6) compared to 0, 5 min (38.3 ± 4.1 and 51.6 ± 5.0, respectively). In conclusion, the fresh diluted camel semen had the lowest viscosity properties after 10 min of incubation with 0.04 mg/mL papain, without compromising sperm motility, viability, acrosome integrity and sperm morphology. [ABSTRACT FROM AUTHOR]

The swine gastrointestinal tract contains a great variety of microbes, forming a complex and dynamic ecosystem. Various internal and external factors (e.g. age, breed and diet) may influence its composition. This study aimed to investigate the gut microbial diversity of German Piétrain boars housed on different deep‐litter bedding materials (regional wood shavings, linen, hemp, spelt husks, and wood shavings) via 16S‐rDNA sequencing. Additionally, short‐chain fatty acids were analysed using gas chromatography. Fresh faecal samples (n = 80) from 40 Piétrain boars were collected twice during the trial. Although it can be assumed that boars ingest bedding orally, no differences in the microbiome composition could be found. The main phyla were Firmicutes and Bacteroides. Acinetobacter was identified as a biomarker for sperm quality differences (total sperm motility) in breeding boars. [ABSTRACT FROM AUTHOR]

This study investigated the effects of storage conditions on the quality of chilled ram semen stored at 4°C for 48 h, comparing aerobic and anaerobic conditions. Ejaculates from INRA180 rams were collected and stored under both conditions, with assessments at 0‐, 24‐, and 48‐h intervals. Various sperm parameters were examined, including motility, velocity, viability, morphology, membrane integrity, and lipid peroxidation. Results showed that storage duration significantly impacted sperm quality, leading to a gradual decline from 0 to 24 h and 24 to 48 h. Notably, after the initial 24 h, progressive motility (PM) and membrane integrity (MI) demonstrated distinct responses to storage conditions. Anaerobic storage consistently improved PM and MI values compared to aerobic storage between 24 and 48 h. Anaerobic conditions also enhanced viability and reduced abnormality at the 48‐h mark. Total motility remained stable throughout storage. Velocity parameters (VCL: curvilinear velocity; VSL: straight velocity and VAP: velocity average path) exhibited differences between the 24‐ and 48‐h intervals, with anaerobic storage resulting in higher VAP and VSL values. Moreover, lipid peroxidation exhibited a progressive increase from 0 to 24 h and 24 to 48 h, independent of storage conditions. Remarkably, anaerobic storage consistently yielded lower lipid peroxidation levels compared to aerobic storage, regardless of storage duration. In conclusion, this study highlights that the anaerobic storage proved advantageous for chilled ram semen quality, particularly after the initial 24 h. [ABSTRACT FROM AUTHOR]

Spermatozoa can experience negative changes when subjected to freezing and thawing, including lowered motility, viability and acrosome response. Herein, the effects of different concentrations of soybean lecithin nanoparticles on cryopreserved Holstein bull semen were examined. Semen was collected, cryopreserved and utilized for sperm kinetic parameter analysis following dilution, equilibration and thawing with 0.5% soybean lecithin (E1), the control extender, and 0.75% (E2), 0.5% (E3), 0.25% (E4) and 0.125% (E5) of lecithin nanoparticles. Results revealed that following dilution, the progressive motility (PM) at E3, E4 and E5 of lecithin nanoparticles was higher (p <.05) than it was for E2. After equilibration, compared to the E1, E2, and E3 values, the PM, vitality, normal morphology, membrane integrity and intact acrosome values at the E5 were consistently greater (p <.05). Comparing the percentages of intact acrosome and membrane integrity at E2 and E3 to E4 and E5, a substantial decrease (p <.05) was seen. Following thawing, the percentage of PM improved at E2 and E5, even though their mean PM values were similar (p >.05) compared to E1, E3 and E4. Vigour and progression parameters of sperm (DAP, DCL, DSL, VAP, VCL, VSL and STR) at E5 were higher (p <.05) than those at E1, E2, E3 and E4. In conclusion, the cryopreserved sperm from Holstein bulls revealed outstanding properties both after equilibration and after thawing with 0.125% lecithin nanoparticles, and they were sensitive to high dosages. [ABSTRACT FROM AUTHOR]

The study investigated midpiece defects in sperm from a 5‐year‐old Brangus bull with a high rate of semen batch rejection, due to morphologically abnormal sperm, with no reduction in sperm kinematics. A comprehensive evaluation was conducted over a 16‐month period, involving 28 ejaculates. Notably, despite the high proportion of midpiece defects (average 37.73%, from 3% to 58%), the study revealed stable sperm production, with no discernible differences in the kinematic data before and after cryopreservation. Electron microscopy identified discontinuities in the mitochondrial sheath, characteristic of midpiece aplasia (MPA). The anomalies were attributed to be of genetic origin, as other predisposing factors were absent. Additionally, the electron microscopy unveiled plasma membrane defects, vacuoles and chromatin decondensation, consistent with previous findings linking acrosome abnormalities with midpiece defects. The findings underscored the necessity of conducting thorough laboratory evaluations before releasing cryopreserved semen for commercialization. Despite substantial morphological alterations, the initial semen evaluation data indicated acceptable levels of sperm kinematics, emphasizing the resilience of sperm production to severe morphological changes. This case report serves as a contribution to the understanding of midpiece defects in bull sperm, emphasizing the need for meticulous evaluation and quality control in semen processing and commercialization. [ABSTRACT FROM AUTHOR]

This study evaluates factors influencing pregnancy rates per artificial insemination (P/AI) and pregnancy loss in Lohi ewes undergoing laparoscopic AI with frozen–thawed semen under sub‐tropical conditions. Data from three experiments comprising ewes (n = 358) of mixed parity (nulliparous; NP and parous; P), various body condition score (BCS) and assigned to long‐term (LTP, 11 days) and short‐term (STP, 5 days) oestrus synchronization regimen across high breeding season (HBS) and low breeding season (LBS) were analysed. Laparoscopic insemination was conducted 54 h post‐sponge removal. Pregnancy diagnosis and loss were evaluated on days 35 and 90 post‐insemination via ultrasonography. Results showed parity significantly influenced P/AI, with nulliparous ewes achieving higher pregnancy ratios than parous ewes (p =.001). BCS significantly influenced P/AI (p <.05), with a quadratic relationship observed between BCS and season (BCS*BCS*Season; p =.07). Progestin treatment did not significantly influence the ratio of pregnant ewes (p =.07). Pregnancy losses were significantly higher during LBS than HBS (p <.05), irrespective of progestin treatment. In conclusion, parity and BCS significantly influenced P/AI, with BCS demonstrating a quadratic association with season. Ewes bred during LBS experienced higher pregnancy losses than HBS, irrespective of progestin treatment. [ABSTRACT FROM AUTHOR]

Cryopreservation is a valuable technique used to assist in the genetic improvement of cultured stocks and provide a continuous supply of good‐quality semen for artificial insemination. Conserving semen by cryopreservation serves several purposes (e.g. artificial reproductive technologies and species conservation) and is also used in the clinical treatment of human infertility. However, the lifespan of cryopreserved semen is influenced by a range of factors, including storage temperature, cooling rate, chemical composition of the extender, the concentration of cryoprotectant, reactive oxygen species, seminal plasma composition and hygienic control. The choice of cryoprotectant is a vital factor underlying the success of animal semen cryopreservation. In this regard, extensive research has been carried out on various cryoprotectants, such as egg yolk, dimethyl sulfoxide, methanol, ethylene glycol and dimethylacetamide. Recent studies have also described the use of a range of new cryoprotectants for cryopreservation, including compounds of plant origin (soy), amino acids, antifreeze proteins, carbohydrates and cyclodextrins. Moreover, semen cryopreservation and storage require the use of liquid nitrogen or ultralow refrigeration methods for both long‐ and short‐term storage. This review summarizes the general methods used for freezing semen and discusses the use of traditional and newly emerging cryoprotectants (permeable and non‐permeable) for the cryopreservation of semen in selected fish and mammalian species. [ABSTRACT FROM AUTHOR]

The aim of this study was to assess the addition of 2% sodium caseinate in a commercial egg yolk‐based medium in frozen ovine semen. Eight Dorper males were used for the study. The ejaculate was divided into two portions and frozen without (G1) or with the addition of 2% sodium caseinate (G2). Kinetic parameters were evaluated using CASA (computer‐assisted sperm analysis), and membrane and acrosome integrity as well as oxidative stress were assessed using flow cytometry. After thawing, a thermoresistance test was conducted at time points T0 and T90. For the fertility test, 100 ewes were inseminated with semen from two rams selected based on in vitro parameters, one with good post‐thaw quality (+70% total motility) and the other with low post‐thaw quality (−55% total motility). For the fertility test, the females were divided into 4 groups for insemination: low‐quality ram without caseinate (GBS = 25) and with caseinate (GBC = 25), and high‐quality ram without caseinate (GAS = 25) and with caseinate (GAC = 25). Regarding the results of sperm kinetics, there was a statistically significant difference in the parameters of average path velocity (VAP) and curvilinear velocity (VCL) between the group frozen with BotuBov and the group with added caseinate. At time point T90, straight‐line velocity maintained a trend (p <.06), with BotuBov® (BB group) being superior to caseinate this time, and in the linearity parameter, caseinate was superior to BotuBov®. Flow cytometry analysis showed no difference between any of the evaluated tests. In the fertility test, there was no statistically significant difference in the pregnancy rate between the BotuBOV® group (23%, 11/48) and the sodium caseinate group (BC group) (33%, 17/52), and no differences were observed in the male versus diluent interaction (p =.70). In conclusion, sodium caseinate supplementation did not influence sperm kinetic parameters and the fertility of sheep. [ABSTRACT FROM AUTHOR]

The effects of an aqueous extract of Scabiosa atropurpurea L. (AES) on the reproduction potential of Queue Fine de l'Ouest rams were evaluated over 9 weeks. Eighteen mature (4–6 years old) rams (52.8 ± 2.6 kg) were divided into three groups. The control (C) group was fed oat hay ad libitum with 700 g of concentrate and the other two groups were fed the same diet supplemented with AES at 1 and 2 mg/kg body weight (AES1 and AES2, respectively). Ram sperm was collected with an artificial vagina (2 × 2 days/week) to evaluate sperm production and quality, antioxidant activity, the adenosine triphosphate (ATP) and calcium concentrations. Sexual behaviour and plasma testosterone concentrations were also investigated. The administration of AES improved sexual behaviour (the duration of contact and the number of lateral approaches). The addition of AES also improved individual spermatozoa motility (C: 71.7% ± 6.3%; AES1: 78.3% ± 4.9%; AES2: 83.8% ± 4.4%), the sperm concentration (C: 5.6 ± 0.36; AES1: 6.4 ± 0.81; AES2: 6.7 ± 0.52 × 109 spermatozoa/mL), the ATP ratio (C: 1 ± 0.08; AES1: 2.1 ± 0.08; AES2: 3.3 ± 0.08) and the calcium concentration (C: 5.6 ± 0.24; AES1: 7.7 ± 0.21; AES2: 8.1 ± 0.24 mmol/L). AES treatment decreased the percentage of abnormal sperm (C: 18.5% ± 1.2%; AES1: 16.2% ± 1.1%; AES2: 14.8% ± 0.94%) and DNA damage (C: 62%; AES1: 27%; AES2: 33%) and was associated with elevated seminal fluid antioxidant activity (C: 22 ± 0.27; AES1: 27.1 ± 1.08 and AES2: 27.5 ± 0.36 mmol Trolox equivalents/L) and plasma testosterone (C: 8.3 ± 0.7; AES1: 11.7 ± 0.4; AES2: 15 ± 0.7 ng/L). In conclusion, our study suggests that S. atropurpurea may be potentially useful to enhance libido and sperm production and quality in ram. [ABSTRACT FROM AUTHOR]

Melatonin is an intracellular antioxidant of sperm membrane that protects the cells from lipid peroxidation. Yet, its role as an antioxidant on semen quality of buffalo bulls is still obscure. The present study was undertaken to assess the effect of exogenous melatonin implant (18 mg/50 kg bodyweight) on post-thaw sperm characteristics, oxidative stress, endocrinological profiles and fertility of buffalo bulls. Six apparently healthy breeding Murrah buffalo bulls were randomly selected at bull farm, Guru Angad Dev Veterinary and Animal Sciences University for the present study and divided into two groups viz. control (n = 3) and melatonin implanted group (n = 3). A total of 120 ejaculates were collected from bulls of both groups (n = 60 each) throughout the study period. Most beneficial effects of melatonin implants were observed during post-implantation period. The percentages of post-thaw sperm total and progressive motility, viability and mitochondrial membrane potential were higher (p < .05) in melatonin implanted buffalo bulls compared to controls during post-implantation period. Following melatonin implantation, MDA production in post-thaw semen was lower (p < .05) in melatonin implanted group than in control group. Plasma melatonin and testosterone concentrations were higher (p < .05) in buffalo bulls implanted with melatonin as compared to their control counterparts. No differences (p > .05) in plasma LH concentrations were observed in both groups. First service pregnancy rate was 43.3% using semen of melatonin implanted bulls and 30.0% with semen of controls (p > .05). Thus, melatonin was able to protect sperm membrane against oxidative damage and improve post-thaw semen quality, thereby resulting in higher fertilizing potential of spermatozoa. [ABSTRACT FROM AUTHOR]

Scrotal surface thermography is a non-invasive method for assessing testicular thermoregulation in stallions; however, few studies have explored the application of this technique concerning the thermal physiology of equine reproductive systems. This study aimed to evaluate the consistency of testicular thermoregulation in stallions over a year using thermography to measure the scrotal surface temperature (SST). Moreover, we assessed the best region for measuring the surface body temperature compared with the SST. Ten light-breed stallions were used in the experiment. Thermographic images of the scrotal and body surfaces (neck and abdomen) were captured. Fresh, cooled and frozen-thawed semen samples were evaluated to verify the impact of thermoregulation on semen quality. Testicular thermoregulation was maintained throughout the year in stallions amidst changes in the external temperature, as evidenced by the weak correlation between the SST and ambient temperature. A lower correlation was observed between the environmental temperature and body surface temperature (BTS) obtained from the abdomen (BTS-A; R = .4772; p < .0001) than with that obtained from the neck (BTS-N; R = .7259; p < .0001). Moreover, both BTS-A and SST were simultaneously captured in a single image. The consistent quality of the fresh, cooled and frozen semen suggests efficient thermoregulation in stallions throughout the year. [ABSTRACT FROM AUTHOR]

To investigate the effect of age in male quail on testicular weight and histology, sexual libido and semen characteristics, a study was performed on 100 quails at 10, 16, 22, 28 and 34 weeks of age. The body and testicular weights were significantly (p <.05) higher at 16 and 22 than at 28 weeks of age. The circumference and diameter of the seminiferous tubules were significantly (p <.05) higher at 28 and 34 than at 10 and 16 weeks of age. Histological evaluation of testicular slices revealed advanced and effective seminiferous tubes as early as 10 weeks, while spermatogenic activity peaked at 16 weeks of age. The highest semen volume, sperm motility and sperm concentration were observed at weeks of age and then decreased gradually with age. At the same time, the testosterone level and libido were significantly (p <.05) higher at 22 than at 10 weeks of age. Furthermore, the age was positive (p <.05), correlated to seminiferous tubule circumference and diameter, and negative (p <.05), correlated to sperm concentration. In conclusion, the age of quail markedly affected the testicular histological structure, libido, testosterone level and semen characteristics. [ABSTRACT FROM AUTHOR]

ProAKAP4 is gaining increasing attention as a potential marker of semen quality in many species, but while there is a commercial kit for assessing proAKAP4 in the domestic cat, there are no publications about its use. The aim of this study was to evaluate the commercial proAKAP4 kit – Cat 4MID® Kit (SPQI – 4BioDx, Lille, France) for the assessment of feline semen. Semen was collected from 54 male cats by urethral catheterization. After a basic semen evaluation (subjective motility, CASA, viability, morphology), proAKAP4 levels in each sample were assessed using the Cat 4MID® Kit according to the manufacturer's protocol or with some modifications related to incubation time, sample storage conditions and number of spermatozoa used. Finally, the Spearman correlation of proAKAP4 concentration and sperm motility parameters was calculated. The most reliable results (acceptable intraassay coefficient of variation) were obtained with an optimized protocol of overnight incubation and isolation of proAKAP4 protein from 1 × 106 spermatozoa stored at −80°C. For fresh semen, there were no significant correlations between proAKAP4 concentration and sperm motility parameters, despite a strong correlation between motility parameters and sperm viability and morphology. A predominant effect of other sperm parameters and highly variable performance of lysis buffer question the usefulness of Cat 4MID® Kit for the assessment of feline semen. For frozen–thawed semen, there was a moderate, negative correlation between proAKAP4 concentration and two CASA parameters, VAP and VSL. As there were no correlations between proAKAP4 concentration in fresh semen and motility parameters in cryopreserved samples, proAKAP4 cannot be used as freezability marker in cats. More studies are needed to establish potential correlation with long‐lasting motility. [ABSTRACT FROM AUTHOR]

This document provides a summary of various studies published in the journal "Reproduction in Domestic Animals." The studies cover a range of topics related to reproductive health in animals, including the role of growth differentiation factor 9 (GDF9) in equine ovary development, the effects of transport-related vibrations on semen quality in stallions, the protective effects of Lactobacillus-conditioned medium on bovine oocyte maturation, and the normal range of testicular diameters in European shorthair cats. Other studies explore simulators for training in artificial insemination, congenital malformations in veterinary medicine, oxygen consumption rates in cryopreserved bovine sperm, and the effects of mito-tempo on cryogenic viability and gene expression in bovine blastocysts. Additionally, the studies investigate the impact of heat stress on reproductive function in dairy cows, age-related changes in mitochondrial metabolism in rabbits, progesterone receptors in goat vaginal tumors, nanoplastics on bovine granulosa cell functions, and the dynamics of polymorph nuclear neutrophils in the oviduct of cows. The document also summarizes articles on the immune response in the bovine oviduct, mucin expression in bovine endometrial cells, lactobacillus treatment for clinical endometritis in dairy cows, interrupted labor in a Van cat, and protein expression in different stages of endometrial tissue. [Extracted from the article]

We evaluated the effects of supplementation with catalase (CAT) and superoxide dismutase (SOD) antioxidants, isolated or combined, on the freezability of collared peccary sperm. Semen aliquots from ten individuals were diluted in Tris‐yolk with or without the presence of antioxidants CAT (200 or 400 IU/mL) and SOD (150 or 300 IU/mL) alone or in combination (CAT: 200 IU/mL + SOD: 150 IU/mL), and then cryopreserved in liquid nitrogen (−196°C). After 1 week, samples were thawed and evaluated for kinetic parameters, membrane functionality and integrity, mitochondrial activity, chromatin integrity, sperm morphology, sperm cell binding capacity and intracellular oxidative levels. Regardless of the use of antioxidants, the treatments did not differ in any of the evaluated parameters, except for the treatment with 300 IU/mL SOD, which showed the highest percentage of static cells (69.1 ± 4.4%) when compared to the group without antioxidant (51.6 ± 3.3%). In general, all samples maintained around 50% of functional membranes, 40% of intact membranes with mitochondrial activity, more than 99% of cells with condensed chromatin, as well as more than 70% of morphologically normal cells. Regarding the binding capacity, the minimum and maximum values found ranged from 33.8 to 194 spermatozoa bound to the perivitelline membrane. Finally, regardless of the presence of antioxidants, all thawed groups showed similar levels of reactive oxygen species (ROS). In conclusion, supplementation with CAT or SOD antioxidants, at the tested concentrations, does not bring benefits to freezability, and the increase in SOD concentration resulted in negative effects on sperm motility in collared peccary semen. [ABSTRACT FROM AUTHOR]

Reduced glutathione (GSH) is a natural redox force immediately consumed by cells to combat toxins. Today's world is shifting towards clean farming practices whence ring‐necked pheasants dependent on croplands are facing threats and sperm‐assisted reproductive techniques become indispensable in preserving this valuable species for game and meat purposes. This study aims at evaluating the cryosurvivability (motility, viability, plasma membrane and acrosomal integrity, DNA fragmentation) and antioxidant capacity (Free radical scavenging activity, LPO detection, Ferric reducing antioxidant power, mitochondrial status) of ring‐necked pheasant semen with GSH supplementation in the extender. Semen collected from ten males was pooled, divided and diluted with Beltsville poultry semen extender (1:5) containing 0.0, 0.2, 0.4, 0.6 and 0.8 mM GSH. Samples were cryopreserved, and sperm quality was evaluated post‐dilution, cooling, equilibration, and freeze–thaw. Free radical scavenging activity, total antioxidant potential, mitochondrial action and lipid peroxidation were assessed at post‐thawing. Sperm motility, plasma membrane and acrosomal integrity, viability and DNA integrity were recorded highest (p <.05) at 0.4 mM GSH compared to other concentrations and control at all the stages of cryopreservation. Similarly, sperm mitochondrial action, antioxidant potential and free radical scavenging capacity were recorded higher (p <.05) with 0.4 mM GSH at post‐thaw compared to other concentrations of GSH and control. Lipid peroxidation was recorded as highest (p <.05) in the control compared to GSH‐supplemented groups at post‐thaw. Hence, it is concluded that GSH at 0.4 mM preserves the quality and ROS scavenging capacity of cryopreserved ring‐necked pheasant sperm as an antioxidant. [ABSTRACT FROM AUTHOR]

The present work was designed for a thorough investigation into the sperm morphology and morphometry of Kurdish stallions. The semen samples were collected from 10 Kurdish stallions. Three preparations from each ejaculate were stained with eosin–nigrosin (EN), Diff‐Quik (DQ) and Rose Bengal (RB). The area, perimeter, length and width of the sperm head as well as tail length and total sperm length were measured. The parameters ellipticity, elongation, roughness and regularity were calculated. The morphology of sperm was also investigated under scanning and transmission electron microscopes. DQ and RB provided more clarified images for examining sperm structures compared to the EN method. The head length, head width, area and perimeter in EN were significantly higher than those in DQ and RB (p ≤.05). Furthermore, the difference in head width, head area and head perimeter between DQ and RB was not significant (p ≥.05). The tail length and total sperm length in all methods were close together (p ≥.05). The highest percentage of normal sperm was seen in DQ and RB methods (82.55 ± 2.88 and 88.31 ± 5.19) respectively. The highest values for ellipticity, elongation and regularity were found in RB, whereas the highest value for roughness was measured in EN. Tail defects including coiled tails, and folded midpieces were the most frequent. Scanning electron microscope revealed two types of head shapes: heads with round anterior border, and heads with flat anterior border. The results indicated that despite the routine use of EN for morphological assessment of stallion sperm, RB and DQ can be considered for more clarified details of sperm structure including acrosome and midpiece. Furthermore, the Kurdish stallion sperm has morphometric traits in the normal range established for stallions; yet, some traits were larger than those reported for other breeds. It seems that the sperm of the Kurdish stallion has a longer head and tail in comparison with other horse breeds. [ABSTRACT FROM AUTHOR]

Electroejaculation (EE) represents the main technique for semen collection from domestic and wild animals independently of libido. However, the technique is associated with intense involuntary muscle contractions, vocalization, ataxia and lying down, caused by the electric stimulation of the nerves in the caudal epigastric region. These clinical manifestations represent important indicators of discomfort. In this context, the objective of this study was to evaluate two protocols of local anaesthetic blockade and two anatomical access for pharmacological desensitization of the caudal epigastric innervation as alternatives to promote comfort and reduce stress associated with EE in rams. For the study, four clinically healthy Dorper rams were selected. All animals were subjected to a design consisting of five semen collection treatments (n = 3 collections per treatment): T1—control, conventional EE without local anaesthetic blockade; T2, EE with ventral blockade (VB) of epigastric innervation using lidocaine hydrochloride 2%; T3, EE with VB of epigastric innervation using a combination of lidocaine hydrochloride 2% and fentanyl citrate; T4, EE with blockade of epigastric innervation through the perineal access using lidocaine hydrochloride 2%; T5, EE with blockade of epigastric innervation through the perineal access using a combination of lidocaine hydrochloride and fentanyl citrate. Seminal samples resulting from EE were subjectively evaluated for sperm motility and concentration, vigour and volume. Additionally, blood serum samples were collected for quantification of cortisol and creatine kinase (CK) enzyme. Assessments of stress and discomfort were conducted by measuring blood pressure, heart rate (HR) and respiratory rate (RR), as well as observing involuntary muscle contractions, ataxia and animal vocalization. No variations in blood pressure, sperm motility, vigour, CK, and cortisol were observed among the treatments. Individual variations were observed for the occurrence of vocalization (p =.0066), but there were no differences between the groups. Anaesthetic blockades conducted using the combination of lidocaine and fentanyl resulted in a lower incidence of ataxia during EE (p <.0001). It is concluded that the combination of fentanyl citrate and lidocaine hydrochloride results in less discomfort for animals undergoing EE, regardless of the anatomical access used for local anaesthetic blockades. [ABSTRACT FROM AUTHOR]

The use of antioxidants for semen preservation prevents oxidative damage caused by reactive oxygen species (ROS). One of the most promising natural antioxidants is resveratrol, a phytoalexin derived from plants, grapes, berries, peanuts and red wine. To evaluate the effect of resveratrol on the quality and redox status of cryopreserved bovine semen. Five bulls were subjected to electroejaculation to obtain 15 ejaculates. Each ejaculate was extended with a tris‐egg yolk‐glycerol‐based medium and divided into six aliquots supplemented with 0 (control), 10, 20, 30, 40 and 50 μM of resveratrol. Semen was frozen with liquid nitrogen vapours. Post‐thawing, motility and kinetics were evaluated using a computer‐assisted sperm analysis (CASA) system, membrane integrity using the hypoosmotic test (HOST), morphology by staining with eosin–nigrosin, sperm vitality by fluorescence microscopy with the SYBR14/IP probes. Total antioxidant capacity (TAC) was evaluated using the ABTS•+ assay and ROS was evaluated using spectrofluorimetry with the H2DCFDA probe. For the statistical analysis linear models were adjusted and means were compared using the Tukey test. All concentrations of resveratrol reduced post‐thawed motility and kinetics of sperm. Supplementation with 40 and 50 μM of resveratrol reduced sperm kinetics, and between 30 and 50 μM of resveratrol alterations in the sperm membrane and morphology were observed. However, using resveratrol at 50 μM increased TAC and at 20 μM, it reduced ROS production of cryopreserved bovine semen. Resveratrol appears to have a dose‐dependent effect in which higher doses produce greater sperm alterations, however, it can increase semen TAC during freezing. It is concluded that resveratrol can increase antioxidant capacity and reduce ROS production in cryopreserved bovine semen. However, its use between 10 and 50 μM reduces post‐thawing semen quality. [ABSTRACT FROM AUTHOR]

This study aimed to determine the effects of Coenzyme Q10 (CoQ10) in the freezing medium on functional and oxidative stress parameters and in vitro fertilization (IVF) rate of buffalo sperm. Collected samples were relocated to the laboratory for initial evaluation, gentle dilution in extenders, cooling (4°C, 2 h), equilibration (4°C, 4 h), packaging (straws, 0.5 mL), programmable freezing, and thawing (37°C, 30 s). Statistical analysis depicted that adding CoQ10 (100 μM) in a freezing medium caused a significant augmentation in total motility (%), average path, and straight‐line velocities (μm/sec) of buffalo sperm than control. Adding CoQ10 (100 μM) improved sperm progressive motility, rapid velocity, and functional parameters (%) compared to the control and 10 μM of CoQ10. Moreover, CoQ10 in a freezing medium caused a significant augmentation in seminal plasma catalase (U/mL) and glutathione reductase (GSH; nmol/109) at 100 μM than control and other treatments. CoQ10 inclusion (100 μM) ameliorates seminal plasma superoxide dismutase (U/mL), glutathione‐S‐transferase (GST; nmol/mL/min) fructose (μg/mL), and ATP (nmol/million) than control. Furthermore, CoQ10 at 100 μM improved seminal plasma glutathione peroxidase (μM) levels than control, 10 μM, and 20 μM. Lastly, hydrogen peroxide (H2O2; nM) production was significantly lower at 100 μM than at control and 10 μM. CoQ10 (100 μM) caused a significant augmentation in the un‐capacitated pattern followed by a reduction in the capacitated pattern, and apoptosis‐like changes (%) than control, and other treatments, whereas viability was increased than control and other treatments. CoQ10 (100 μM) significantly improved the IVF rate in comparison with control, CoQ10 at 10 μM, and 20 μM groups. In conclusion, the addition of CoQ10 (100 μM) in the freezing medium can improve the quality and in vitro fertility of post‐thawed buffalo semen via its antioxidative effect. Further studies are needed to evaluate the effect of CoQ10 on the in vivo fertility of buffalo bull semen. [ABSTRACT FROM AUTHOR]

While sperm migrate within the reproductive tract of cows experiencing negative energy balance (NEB), they come into contact with elevated concentrations of non‐esterified fatty acids (NEFA). For this reason, this study aimed to investigate the effects of three different NEFA – palmitic acid (PA), stearic acid (SA), and oleic acid (OA) – on bovine sperm motility, kinetic parameters, oxidative status, and morphology. Frozen thawed semen samples from Bos taurus bulls were incubated with varying concentrations of each fatty acid, and the sperm's characteristics were analysed at different time points. Computer‐Assisted Sperm Analysis (CASA) was employed to assess sperm motility and kinetic parameters. Concurrently, the production of the reactive oxygen species (ROS) and total antioxidant capacity were measured to determine the oxidative status. Additionally, sperm morphology was evaluated. In Experiment 1, different concentrations of PA did not show significant effects on total motility, progressive motility, or any kinetic parameters analysed. Similarly, PA did not have a significant impact on the oxidative status or sperm morphology. In Experiment 2, SA at various concentrations did not lead to significant changes in total motility, progressive motility, or any kinetic parameters evaluated. Furthermore, SA did not affect oxidative status or sperm morphology. In Experiment 3, the concentrations of OA used did not result in significant changes in total motility, progressive motility, or any kinetic parameters studied. Likewise, OA did not induce any alterations in oxidative status or sperm morphology. Overall, the results from all three experiments indicate that PA, SA and OA, at the in vitro conditions and tested concentrations, do not exert detrimental effects on bovine sperm function and morphology. These results provide insights that contribute to our understanding of how fatty acids can impact the reduction of fertility rates in cows facing NEB. This, in turn, lays the foundation for additional critical investigations in this area. Further studies are necessary to validate these findings in vivo. [ABSTRACT FROM AUTHOR]

The current study aimed to investigate the impact of nano‐formulations of clove bud ethanolic extract (CBENF) in the extender on sperm characteristics, antioxidant capacity, oxidative biomarkers, enzymatic activity, apoptosis and fertility of post‐thawed rabbit semen. Twelve mature male rabbits semen samples were pooled and cryopreserved in a Tris‐egg yolk‐based extender containing varying concentrations of CBENF (0, 25, 50, 75 and 100 μg/mL). After the equilibration and freezing–thawing process, CBENF (100 μg /mL) significantly enhanced progressive motility, viability and membrane integrity. Conversely, sperm abnormality was significantly reduced by CBENF supplementation. Total antioxidant capacity was increased in the post‐thawed sperm medium, while nitric oxide and malondialdehyde were decreased in all CBENF concentrations. The lactic dehydrogenase and caspase‐3 activities were decreased, whereas the number of live spermatozoa with an intact acrosome was increased in all CBENF concentrations. Conception rate and litter size per doe were higher in doe rabbits inseminated with semen supplemented with 100 μg CBENF/mL than un‐supplemented group (76% vs. 52% and 8.4 vs. 7.7/doe), with no statistical differences. These findings suggest that supplementing rabbit extenders with 100 μg of CBENF/mL could be an effective strategy for enhancing freeze–thawing rabbit sperm attributes and fertility. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the morphological, functional and molecular changes in frozen–thawed ram sperm using an extender containing different concentrations of hydrated carbon 60 fullerene (C60HyFn), a nanotechnological product. Semen taken from each of the seven Akkaraman rams were pooled. Semen collection was done twice a week and it continued for 3 weeks. Each pooled semen sample was divided into six equal groups and diluted with tris + egg yolk extender including 0 (control), 200, 400, 800 nM, 1 and 5 μM concentrations of C60HyFn at 37°C. They were then frozen in liquid nitrogen vapour at −140°C, stored in liquid nitrogen container (−196°C) and thawed at 37°C for 25 s before analysis. In comparison with control, C60HyFn addition prior to freezing procedure provided significant increases in total and progressive motility rates, glutathione peroxidase, catalase activities and percentage of highly active mitochondria, and significant decreases in dead and abnormal sperm rates, lipid peroxidation, caspase‐3 and DNA fragmentation levels in frozen–thawed ram semen. When compared to control, C60HyFn supplementation significantly down‐regulated the expression levels of miR‐200a and KCNJ11, and significantly up‐regulated the expression levels of miR‐3958‐3p (at the concentrations of 200, 400, 800 nM and 1 μM), CatSper1 (at the concentrations of 200, 400 nM and 5 μM), CatSper2 (at the concentrations of 1 and 5 μM), CatSper3 (at the concentrations of 200, 400 nM, 1 and 5 μM), CatSper4 (at all concentrations), ANO1 (at the concentrations of 800 nM, 1 and 5 μM) and TRPV5 (at the concentrations of 200, 400 and 800 nM). The addition of C60HyFn had no effect on global DNA methylation rates. As a result, C60HyFn supplementation to ram semen extenders may be beneficial in reducing some of the functional, structural and molecular damages in sperm induced by the freeze–thawing procedure. [ABSTRACT FROM AUTHOR]

Sperm capacitation is a crucial step towards the acquisition of fertilizing capacity. Despite the attempts to mimic the in vivo situation, there is still a lack of standardization in vitro techniques. Bicarbonate and serum albumin (BSA) are routinely used, although controversial results are reported regarding the optimal concentration of each compound. In addition, whether caffeine is needed on in vitro capacitation media in boar sperm remains to be elucidated. Here, 18 boar commercial artificial insemination doses were used to test different concentrations of bicarbonate (19, 37 or 56 mM) in experiment 1, BSA (1.5, 3, 4.5 mg/mL) in experiment 2 and the presence or absence of caffeine (5.15 mM) experiment 3. We analysed at 0, 30 and 120 min of incubation at 38.5°C, 5% CO2: Total motility (TMOT), membrane integrity (VIAB), acrosomal exocytosis (rAcro; H33342/PI/PNA), capacitation status (chlortetracycline staining CTC) and mitochondrial membrane potential (JC‐1). The higher concentrations of bicarbonate (37 and 56 mM) decreased TM and VIAB (p <.01) but increased rAcro (p <.01) after 120 min of incubation compared to the fresh control. In contrast, only the BSA concentration of 3 mg/mL reduced the VIAB at 120 min, but all the concentrations tested increased the average of JC‐1 and decreased TM (p <.01) throughout incubation compared to the fresh control. Finally, in experiment 3, when boar sperm were incubated in the capacitating media with bicarbonate, BSA and with or without caffeine, the capacitated pattern measured by the CTC technique and rAcro increased after 120 min of incubation (p <.01) compared to fresh control, either in the presence or in the absence of caffeine. In summary, our results suggested that the combination of capacitating components, like bicarbonate and BSA, contributed to increasing the proportion of capacitated boar spermatozoa, mitochondrial membrane potential as well as acrosomal exocytosis. However, caffeine did not significantly influence in vitro sperm capacitation in this species. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the relationship between the levels of insulin‐like growth factor‐1 (IGF‐1) in serum and seminal plasma and the characteristics of semen in Beetal bucks (Capra hircus). A total of 12 adult Beetal bucks were involved in the study, with each buck providing six ejaculates collected using a standard artificial vagina (n = 72 total). Only qualified semen samples (volume of 0.7 mL, a mass motility rating of 3+ or higher on a 0–+ scale, and individual progressive motility of 80% or more) divided into three fractions were processed for estimation of IGF‐1 and other seminal parameters like motility, viability, acrosome integrity, sperm abnormality and superoxide dismutase (SOD) activity. The first and second fraction were diluted and extended with Optixcell extender (1:15 ratio). The first ejaculate fraction was processed for studying fresh semen parameters and the second fraction was cryopreserved for evaluating frozen semen parameters. French mini straws (0.25 mL) were used for semen filling, and polyvinyl alcohol powder of different colours was used for sealing the extended semen. The third fraction of each ejaculate was centrifuged at room temperature (1100 × g for 7 min) to separate the seminal plasma. Additionally, blood samples were taken from each buck on the same day as semen collection, resulting in a total of 36 blood samples. The results revealed a significant positive correlation (r =.4243; p <.05) between the concentration of IGF‐1 in both serum and seminal plasma of the Beetal bucks. Furthermore, the concentration of IGF‐1 in serum showed significant positive correlations with sperm viability (r =.554; p <.05), acrosome integrity (r =.527; p <.05), post‐thaw sperm motility (r =.407; p <.01), post‐thaw sperm viability (r =.426; p <.01) and post‐thaw acrosome integrity (r =.333; p <.05). However, it had a significant negative correlation with SOD activity in fresh semen (r = −0.458; p <.01). Moreover, the concentration of IGF‐1 in seminal plasma demonstrated significant positive correlations with individual progressive motility (r =.341; p <.05), sperm viability (r =.527; p <.05), acrosome integrity (r =.539; p <.05), sperm plasma membrane integrity (r =.464; p <.05), post‐thaw sperm motility (r =.644; p <.01), post‐thaw sperm viability (r =.643; p <.01), post‐thaw acrosome integrity (r =.487; p <.01) and post‐thaw sperm plasma membrane integrity (r =.521; p <.01). Additionally, it showed a significant negative correlation with SOD activity in both fresh semen (r = −0.714; p <.01) and frozen semen (r = −0.558; p <.01) of Beetal bucks. Based on these findings, IGF‐1 in seminal plasma can be considered as a potential biomarker for the selection of bucks for breeding purposes. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the effect of antioxidant supplementation in diluted semen from Muscovy drakes after the induction of oxidative stress (OS) on the sperm motility, kinematic parameters and biochemical markers – lipid peroxidation (LPO) levels and total glutathione (tGSH) concentration. The pooled semen was distributed equally into three parts, diluted (1:3 v/v) with IMV Canadyl, HIA‐1 or AU, and stored at 4°C for 6 h. Later, the semen was equilibrated at 20–25°C for 15 min, and divided in Eppendorf tubes. The sperm samples (final concentration of 50 × 106 sperm cells/mL per sample) were incubated at 37°C for 30 min in the absence (−) or presence (+) of 0.1 mM FeSO4 + 0.5 mM H2O2 (Fenton system) and the following combinations of antioxidants: ascorbic acid + Trolox (A + T); ascorbic acid + Desferal (A + D); Trolox + Desferal (T + D) and ascorbic acid + Trolox + Desferal (A + T + D), all of them in a final concentration of 0.1 mM. Thus, the total number of samples was 30 and in each one, the sperm motility, velocity parameters, LPO and tGSH were determined. The motility and kinematic parameters of the diluted semen with added antioxidants were restored by up to 20% after inducing OS via the Fenton reaction. Dual combinations of antioxidants (A + T, A + D, and T + D) lowered LPO levels, but not equally across different extenders. After the induction of OS, the tGSH levels in diluted semen with IMV–Canadyl were not affected by the added antioxidants. Whereas antioxidant combinations in diluted semen with HIA‐1 or AU had a beneficial effect and partially restored tGSH levels. In conclusion, the results showed that the extender IMV–Canadyl is well balanced and protected the Muscovy semen under OS conditions, while the other two extenders HIA‐1 and AU can be improved by adding antioxidants. [ABSTRACT FROM AUTHOR]

In this study, the relationships between post‐thaw bull sperm characteristics and hyperketonemic conditions after coincubation with cow plasma or media were determined to investigate if such a condition could affect bull sperm characteristics. Two experiments were conducted. In experiment 1, blood samples were collected from 31 cows to prepare plasma. Cows were independently categorized into two groups according to plasma β‐hydroxybutyrate (BHB) concentrations (above or below 1.2 mM). Thawed bull semen was diluted and incubated with diluted plasma; motility parameters were evaluated using Computer Assisted Semen Analysis (CASA). In experiment 2, a pooled sample of thawed semen was diluted and divided into three aliquots: without BHB (control) and treated with either 1.2 mM (1.2) or 3 mM (3) BHB. In addition to motility, flow cytometric analyses were carried out. In experiment 1, the overall motility decreased significantly in plasma containing high (≥1.2 mM) BHB compared to plasma containing low (<1.2 mM) BHB. In experiment 2, the overall motility tended to be lower in BHB (3 mM)‐supplemented samples. The supplementation of 3 mM BHB increased the proportion of live superoxide‐positive sperm and sperm with high mitochondrial potential, while the DNA fragmentation index decreased. [ABSTRACT FROM AUTHOR]

Sperm preservation is a well‐established technique in reproductive biotechnology that is widely used to maintain the genetic quality of male individuals. However, there are several factors during the preservation process that can affect the vitality, functionality, and quality of sperm, thereby reducing their fertility potential after thawing. One of these factors is the synthesis of high levels of oxidative stress (OS) during semen preservation, which can have detrimental effects on sperm health and functionality. To counter the negative impact of OS on sperm, researchers have explored the supplementation of several exogenous antioxidants in the extenders used to preserve ram sperm. This approach has shown promising results in improving sperm health, functionality, and fertility potential in ram. Additionally, the preservation process can induce modifications in the ram sperm proteome. By employing targeted proteomics techniques, researchers have been able to identify and modify specific proteins in cryopreserved ram sperm, potentially offering further improvements in the quality of the cryopreserved ram sperm. In summary, this review provides a comprehensive overview of the antioxidants and targeted proteomics modifications that have been investigated for enhancing ram sperm preservation. These advancements aim to mitigate the negative effects of OS and optimize the techniques used in preserving ram sperm. [ABSTRACT FROM AUTHOR]

Sexed semen facilitates additional female calf production for the expansion of a herd at a faster rate and also curtails the surplus production of unwanted male calves. A study was conducted to evaluate the performance of sexed semen in indigenous Tharparkar cows based on 114 artificial inseminations (AI) performed at natural oestrus using two protocols i.e., single AI (n = 48) and double AI (n = 66). Overall, the first service conception rate (CR) was significantly higher in double (53.0%) than single (33.3%) AI protocol. The odds ratio of conception rate in double AI was 2.26 (χ2 = 4.4, df = 1, p =.04) with respect to single AI. The time that elapsed since the detection of oestrus to insemination was also analysed. In a single AI protocol, the CR was higher (p <.05) at 16 h (54.6%) than insemination at 8 h (27.0%) following the onset of oestrus. Yet, the CR using double AI protocol did not differ (p =.73) significantly when AIs were performed either at 8 h and 24 h (51.9%) or 16 h and 24 h (57.1%) post onset of oestrus. Besides, like the single AI protocol, the parity of the animals also influenced the CR, being higher in heifers (n = 22) than those of parous (n = 92) cows (72.73 vs. 40.43%, χ2 = 7.48, df = 1, p =.006) in the present study. The odds ratio of conception in heifers was 3.93 with respect to parous cows. Overall, the birth of female calf was 91.7%. In conclusion, the present study indicates a future promise of the sexed semen for the production of more female offspring from Tharparkar cattle. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the effect of supplementing bovine semen freezing extender with different concentrations of iodixanol on post‐thaw sperm characteristics. Six ejaculates of three Nellore bulls were pooled and diluted in commercial extender (BotuBov®) and then divided into 4 groups: control group (without adding iodixanol); groups G1.5, G3, or G6 according to the concentration of iodixanol solution (RedCushion®). After dilution, the samples were cooled and frozen. Post‐thaw semen evaluation included sperm motility by CASA immediately after thawing and after 60 min of incubation at 37°C, flow cytometry analysis for integrity of plasma and acrosomal membranes, membrane destabilization and translocation of phosphatidylserine, mitochondrial membrane potential, and formation of intracellular anion superoxide (O2−), hydrogen peroxide (H2O2), and membrane lipid peroxidation. The group G6 presented significantly higher (p <.05) total and progressive motility, percentage of plasma and acrosomal membrane integrity, and H2O2 than control and group G1.5. Furthermore, group G6 showed lower (p <.05) lipid peroxidation than control. In addition, regardless of the concentration used, the percentage of spermatozoa without phosphatidylserine translocation was higher (p <.05) in all iodixanol supplemented groups. In conclusion, iodixanol supplementation preserved the motility and integrity of sperm membranes during cryopreservation and protected against lipid peroxidation. [ABSTRACT FROM AUTHOR]

The identification of different morphometric patterns of spermatozoa serves as a basis for improving our understanding of the diversity in an ejaculate and to relate them to the potential fertility of males. In this study, we aimed to examine the semen subpopulation structure, following dilution in semen of extenders, using a mathematical approach a possible application to fertility analyses. Ten sexually mature Bos taurus bulls were randomly allotted to one of three groups: (1) Tris–citric acid–egg yolk extender (Tris‐EY); (2) commercial egg yolk extender OptiXcell® and (3) commercial egg yolk extender Triladyl®. The results showed significant differences (p <.05) between extenders in terms of values for head size and head shape variables of individual sperm, indicating an influence of extender composition. Sperm head width was found to significantly differ (p <.05) according to the extender, decreasing in the following order: OptiXcell® (4.836 ± 0.017 μm), Triladyl® (4.695 ± 0.012 μm) and Tris‐EY (4.638 ± 0.010 μm). Principal component analysis allowed us to identify two subpopulations in OptiXcell®, and three subpopulations were each found in Triladyl® and Tris‐EY. Overall, we observed significant differences between sperm subpopulations within each extender (p <.05), with differences in sperm head size and shape between bovine species that can be related to functionality and fertility capabilities. [ABSTRACT FROM AUTHOR]

Appropriate stallion semen handling is of great importance in equine artificial insemination (AI) industry. Optimal treatment of AI doses is aiming for best sperm preservation by excluding strong environmental influences and adding media that favour sperm survival. One method widely used in stallion sperm handling is the rotation of liquid‐preserved semen samples on roller benches during storage. As previous studies in boars give rise to the fact that rotation should not be considered beneficial for spermatozoa anymore, the present study investigated the influence of continuous rotation of diluted stallion AI portions on sperm quality. Ejaculates (n = 15) were collected at a German AI centre and diluted with the two extenders EquiPlus and Gent (Minitüb GmbH) to a final concentration of 50 × 106 sperm/mL. Afterwards, samples were placed separately on roller benches at 5°C in the dark, obtaining a rotation frequency of 5 revolutions per minute (rpm) and 36 rpm for four consecutive days following a split‐sample design. Both groups were analysed daily in comparison to a control group (0 rpm) with an extended spectrum of spermatological methods including computer‐assisted sperm analysis and flow cytometry. Statistical analyses were based on the calculation of generalized linear mixed models for each sperm parameter. The research revealed a decrease in sperm quality parameters of rotated samples compared to non‐rotated control groups, visible in total sperm motility (p <.001), decreased thermo‐resistance (p <.01) and a drop in pH (p <.001). Interestingly, no differences (p >.05) were detected between rotation frequencies of 5 and 36 rpm. We conclude that the fertilizing capacity of stallion semen was negatively affected by rotation during storage in vitro, irrespective of the rotation frequency. Further studies need to investigate whether field fertility in horses is similarly affected by semen rotation on roller benches in vivo. [ABSTRACT FROM AUTHOR]

Low‐density lipoproteins (LDL), quercetin (Q) and resveratrol (R) have been used for sperm preservation to improve sperm quality in different species. To evaluate the effects of LDL, Q and R during the cooling of boar semen. Fifteen boar semen samples were diluted in a BTS extender supplemented with the treatments: LDL at 6%, Q at 10 μM (Q10), 30 μM (Q30) and 50 μM (Q50), or R at 10 μM (R10), 30 μM (R30) and 50 μM. A control without supplementation was included. The semen was stored by cooling at 16°C for 96 h. Every 24 h, sperm motility and kinetics were evaluated using a computer‐assisted sperm analyzer (IVOS). At 24 and 96 h of cooling, functional membrane integrity and mitochondrial membrane potential (ΔΨM) of sperm were evaluated by the hypoosmotic swelling test (HOST) an flow cytometry with JC‐1 probe, respectively, LDL improved progressive motility of sperm during cooling. Likewise, LDL increased average path velocity (VAP) and straight‐line velocity (VSL) and/or curvilinear velocity (VCL) during the first 48 h of cooling. The use of Q between 10 and 30 μM caused a reduction in total motility, progressive motility and amplitude of the lateral head displacement during the entire cooling period, as well as a decrease in VAP, VSL and VCL at 96 h of cooling. LDL, Q10, Q30 and Q50 modulated mitochondrial activity by reducing high‐ΔΨM sperm at 0 and 96 h of cooling. During the cooling of the boar semen prior to artificial insemination, the parameters of sperm quality that could influence fertility decrease; however, the inclusion of antioxidants and additives that protect the plasma membrane, such as LDL, could mitigate the damaging effects on spermatozoa. It is concluded that LDL can improve the motility and kinetics of boar semen during cooling while it could modulating the sperm's mitochondrial activity. On the contrary, Q could alter the motility and kinetics of boar sperm during the cooling period. [ABSTRACT FROM AUTHOR]

To improve accuracy in evaluating stallion ejaculates, an antibody‐based, flow cytometric assay for the detection and identification of leukocyte subpopulations (CD4‐, CD8‐, CD21‐, CD172a‐positive cells) in stallion semen (n = 12) was established. For establishment of the assay, native semen was supplemented with blood leukocytes (control: 20% leukocytes, 80% sperm cells) and analysed by flow cytometry. Adding antioxidants (ascorbic acid and butylated hydroxytoluol) to semen immediately after collection inhibited rapid death of lymphoid cells in sperm leukocyte mixtures. In control set‐ups, 27.85 ± 5.7% of events were positive for CD4, CD8, CD21 or CD172a, while in native semen samples, leukocytes were scarce (0.114 ± 0.134%). The most abundant leukocyte subpopulation in semen was of lymphoid origin (CD4‐positive cells [0.015 ± 0.02%]), whereas CD21‐positive cells (B cells; 0.001 ± 0.001%) were virtually absent in ejaculates of fertile stallions. This presented flow cytometric assay for the detection and identification of different leukocyte population in equine antioxidant‐treated ejaculates can be used as an additional tool for spermatological examination in stallions. [ABSTRACT FROM AUTHOR]

Anti-muellerian hormone (AMH) concentrations during pregancy and lactation in dogs S. Arlt 1; J. Hinderer 2; L. Riege 2; J. Leber 2; E. Müller 3; C. Weber 3; S. Müller 3... 1 Clinic of Reproductive Medicine, Vetsuisse Faculty, University of Zurich, Zurich, Switzerla... The anti-Müllerian hormone (AMH) is a glycoprotein secreted mainly by granulosa cells in pre-antral and small antral follicles in female dogs. In conclusion, conservative treatment with splint application can be considered as a non-invasive treatment of kittens affected by thorax deformity. CCO 19 Conservative treatment of flat thorax in two littermate kittens: A case report J. Fusi; J. Bassi; M. Veronesi Department of Veterinary Medicine and Animal Sciences, Università degli Studi di Milano, Lodi... Three 18 days-old kittens were referred because of severe dyspnea and thorax flattening of one subject. Two weeks after splint removal, both kittens were healthy, and showed normal behaviour of 45 days old kittens, including playing, running, jumping, even though kitten A still displayed a slight deformation of the thorax profile. [Extracted from the article]

The use of assisted reproductive techniques, such as chilled semen, contributes to the maintenance and genetic improvement of canine breeding. The INRA‐96 extender is a commercially available, chemically defined medium that was initially developed for the preservation of equine semen and exhibits preservation potential in the canine species. This research aims to evaluate the INRA‐96 extender as an alternative for the short‐term preservation of canine semen in terms of sperm quality parameters such as motility and kinetic parameters, integrity and functionality of the plasma membrane in fresh and chilled–rewarmed samples, as well as the sperm‐binding ability using the perivitelline membrane of the chicken egg as an indicator of the fertilizing capacity of the preserved semen. A total of 18 ejaculates from 9 French bulldogs (two ejaculates per dog) were collected and divided into two aliquots that were diluted in Tris‐egg yolk 20% (control) or INRA‐96 to a final concentration of 100 × 106 sperm/mL. Samples were refrigerated in a biological incubator at 5°C and evaluated at 0, 24 and 48 h time points. Comparing the two treatments after 48 h of refrigeration, both extenders showed similar values (p <.5) for the majority of kinetic parameters, with the INRA‐96 group promoting a total motility of 88.1 ± 2.9%. In addition, the morphology, integrity and functionality of the plasma membrane were preserved above 70% in this group. Dilution with INRA‐96 also provided a significantly higher amount of sperm bound (256.2 ± 21.1) to the perivitelline membrane of the egg yolk compared to the sperm‐binding rates (p <.05) achieved at the use of Tris‐egg yolk (215.2 ± 21 bound spermatozoa) at 48 h. Our study proved similar functional properties of dog sperm cells treated with INRA‐96 in comparison to commonly used home‐made Tris‐based extender during short‐time storage. [ABSTRACT FROM AUTHOR]

The use of α2‐adrenergic agonists in association with urethral catheterization has been used as a technique for pharmacological semen collection in cats. The mechanism of action of this drug is the stimulation of adrenoreceptors in the vas deferens, which results in ejaculation. While medetomidine is the α2‐agonist most commonly used in studies, ejaculation with the use of dexmedetomidine associated with ketamine has been effective, but with variable results. Therefore, further studies regarding the methodology of use are required to obtain better seminal quality. This study aimed to compare two pharmacological semen collection times after the association of dexmedetomidine (30 μg/kg, IM; Dormitor®, Zoetis), ketamine (5 mg/kg, IM; ketamine, Vetnil) and urethral catheterization using a tomcat probe (0.8 mm × 1.00 mm × 11 cm). The collections were divided into two experimental groups: G10 (N = 8; urethral catheterization after 10 min of anaesthesia) and G15 (N = 8; urethral catheterization after 15 min of anaesthesia). The ejaculates were evaluated for ejaculate volume, sperm concentration, morphology and kinetics using the CASA system. To compare the groups, the t‐test and the Mann–Whitney U‐test were used with a significance level of 5%. It was identified that ejaculate volume (G10: 22.62 ± 2.13 vs. G15: 26.81 ± 1.55; p <.001) and sperm concentration (G10: 48.10 × 106 ± 17.84 vs. G15: 90.18 × 106 ± 19.35; p <.001) was higher in G15 than in G10 and had a lower percentage of minor defects than G10 (G10: 3.12 ± 2.41 vs. G15: 1.00 ± 1.19; p =.043). Regarding the kinetic parameters, the results of G15 were better for total motility—TM (G10: 67.00 ± 10.33 vs. G15: 81.87 ± 7.99; p =.006) and faster cells—RAPID: (G10: 55.00 ± 16.63 vs. G15: 74.25 ± 11.94; p =.019); whereas a higher proportion of cells with slow speed—SLOW were seen in G10 (G10: 31.00 ± 12.07 vs. 17.12 ± 7.53; p =.015). Based on these findings, we suggest that collection via urethral catheterization should be performed 15 min after the application of ketamine‐associated dexmedetomidine to obtain a better‐quality ejaculate. [ABSTRACT FROM AUTHOR]

The Indian red jungle fowl population is decreasing in its natural habitat. Its conservation through semen cryopreservation with sufficient live sperm recovery rate is requisite where ascorbic acid could play significant role to mitigate cryo‐incited injuries. The objective was to elucidate the effect of ascorbic acid on freezability of Indian red jungle fowl sperm. Pooled semen was aliquoted and diluted (1:5) with red fowl extender having ascorbic acid: 0.0 (control), 1.0, 2.0 and 4.0 mM. Diluted samples were cryopreserved and semen quality was assessed at post‐dilution, cooling, equilibration and freeze–thawing stages. Sperm metabolic status, antioxidant potential and lipid peroxidation were studied at post‐dilution and freeze–thawing. Sperm motility did not differ (p >.05) in experimental extenders and control at post‐dilution and cooling; however, it was recorded higher (p <.05) with ascorbic acid at 2.0 mM compared with other levels at post‐equilibration and post‐thawing stage. Sperm viability, plasma membrane and acrosome intactness were recorded higher (p <.05) with 2.0 mM ascorbic acid compared with other concentrations of ascorbic acid at all stages of cryopreservation. Sperm metabolic status and antioxidant potential were recorded higher (p <.05), while lipid peroxidation was recorded lowest (p <.05) with 2.0 mM ascorbic acid compared with 1.0, 4.0 mM and control. In conclusion, ascorbic acid at 2.0 mM in red fowl extender improves quality, metabolic status and antioxidant potential of frozen Indian red jungle fowl semen through ameliorating lipid peroxidation. [ABSTRACT FROM AUTHOR]

In a previous study, our group detected the cholecystokinin (CCK) protein in the porcine oviduct. This fact, together with the involvement of CCK in the regulation of sperm protein tyrosine phosphorylation by the modulation of HCO3− uptake (in mice and humans) suggests a role for CCK during sperm capacitation. Therefore, on the one hand, the expression of CCK receptors (CCK1R and CCK2R) on boar testes has been investigated and probed; on the other hand, boar spermatozoa (from seminal doses of 1‐day and 5‐day storage) were exposed to different concentrations of CCK (0‐control, 25 or 50 μM) in a medium supporting capacitation supplemented with 0, 5 or 25 mmol/L of HCO3− for 1 h at 38.5°C. Sperm motion (total and progressive motility), kinetic parameters, viability, acrosome status, and mitochondrial activity were determined. No differences between groups (0, 25 or 50 μM of CCK) were observed when HCO3− was absent in the media (p >.05). However, the results showed that when the media was supplemented with 5 mmol/L HCO3− in 1‐day seminal dose storage, the linearity index (LIN, %), straightness index (STR, %) and oscillation index (WOB, %) (sperm kinetics parameters) increased in the presence of CCK regardless the concentration (p <.05). Nevertheless, CCK in sperm from 5‐day storage only increased the WOB parameter in comparison to the control (p <.05). Furthermore, the average amplitude of the lateral displacement of the sperm head (ALH, μm) and curvilinear velocity (VCL, μm/s) decreased when CCK was present, depending on its concentration and sperm aging (1‐day vs. 5‐days) (p <.05). In the case of the media supporting capacitation supplemented with 25 mmol/L HCO3−, any differences were observed except for sperm viability in the 5‐day seminal doses, which increased in the 50 μM‐CCK group compared to the control (p <.05). In conclusion, these data suggest an implication of CCK protein during sperm capacitation under low bicarbonate concentration increasing the sperm linear trajectory. [ABSTRACT FROM AUTHOR]

The use of antibiotics in semen extenders can contribute to the development of antibiotic resistance. The objective of the study was to evaluate epsilon‐polylysine (Ɛ‐PL) as a substitute for antibiotics in the buffalo semen extender. For this, 20 semen ejaculates were collected from four Murrah buffalo bulls. Each ejaculate was divided into three equal aliquots and extended into an egg yolk‐based semen extender containing either antibiotics (strepto‐penicillin) or different concentrations of Ɛ‐PL (0.64 and 1.28 g/L) to make the final concentration 80 million sperm/mL and cryopreserved as per the standard procedure. The antibiogram sensitivity test confirmed that Ɛ‐PL is an effective antimicrobial against microbes present in buffalo semen ejaculates. Furthermore, the addition of Ɛ‐PL in the semen extender significantly reduces the colony forming unit (CFU)/mL in cryopreserved semen equivalent to strepto‐penicillin. The sperm motility and kinematic parameters assessed by a computer‐assisted sperm analyser showed that Ɛ‐PL did not inhibit either sperm motility not kinematic parameters of cryopreserved sperm. The flow‐cytometric evaluation of frozen–thawed sperm revealed interesting results. The extender supplemented with Ɛ‐PL protected sperm acrosome and mitochondrial membrane potential greater than the extender supplemented with strepto‐penicillin. Further, Ɛ‐PL reduced significantly the production of superoxide anions from mitochondria during the cryopreservation process. In this way, Ɛ‐PL may be a suitable alternative to antibiotics in semen extenders. In conclusion, Ɛ‐PL at a concentration of 0.64 g/L acts as an effective antimicrobial as well as antioxidant in semen extender for cryopreservation of buffalo sperm. [ABSTRACT FROM AUTHOR]

To the best of our knowledge, no research has been conducted to test the effects of syringic acid (SA) on ram semen freezing within the scope of natural antioxidants added to semen extenders. Therefore, this study had two main objectives. First, to test whether adding SA to ram semen freezing extender has a protective effect and contributes positively to sperm kinetic, plasma and acrosome integrity, mitochondrial membrane potential, lipid peroxidation, oxidant and antioxidant and DNA damage parameters after thawing. Second, it was to determine at what concentration the SA supplemented to the extender could be applied by in vitro studies by preserving the fertilization ability of frozen semen at the highest level. In the study, six individuals of Sönmez rams were used. The semen was collected from the rams using an artificial vagina and pooled. The pooled semen was divided into five different groups and extended with 0, 0.5, 1, 2 and 4 mM SA (control C, SA0.5, SA1, SA2 and SA4, respectively). After dilution, the semen samples were kept at 4°C for 3 h, then loaded into 0.25 mL straws and frozen in liquid nitrogen vapour. The SA1 and SA2 groups were higher plasma membrane and acrosome integrity (PMAI), high mitochondrial membrane potential (HMMP), plasma membrane integrity and motility compared to other groups (p <.05). It was observed that SA supplemented to the Tris extender significantly reduced DNA damage, and the lowest values were obtained especially in the SA1 and SA2 treatments (p <.05). Also, lowest MDA level was determined at the SA1 and this was statistically significant compared to SA4 and C (p <.05). In conclusion, it was revealed that SA added to Tris semen extender at 1 and 2 mM treatment doses increased progressive and total motility and preserved PMAI, plasma membrane integrity, HMMP and DNA integrity. [ABSTRACT FROM AUTHOR]