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The pterostilbene (PT) molecule is a phytoalexin with a reducing effect on reactive oxygen species (ROS) and with a capacity to block lipogenesis. However, the potential reducing effects of PT on equatorial lipid accumulation and ROS have not yet been elucidated for in vitro-derived bovine embryos. The present study evaluated the effects of concentrations of 3, 1, 0.33, 0.11 μM PT, and a vehicle group on the percentage of cleaved embryos, embryos with more than 6 cells, percentage of blastocyst on Day 7 and 8, percentage of transferable embryos on Day 7, the cell count and relative concentration of lipids. In the second experiment, the effects of 0.33 μM PT and a vehicle group within two different O2 environments (5% and 20%) were evaluated for ROS generation and the percentage of Day 8 blastocysts. In the first experiment, no significant differences were found between the treatments with PT and the vehicle group (p > .05) concerning the percentage of cleaved embryos and embryos with more than 6 cells. Lipid reduction was observed in the groups treated with PT versus the vehicle group (p .05). In the second experiment, the O2 concentration did not significantly affect ROS generation (p > .05); however, the groups treated with PT (0.33 μM) had a reduction in ROS (p

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by a mutation that expands the polyglutamine (CAG) repeat in exon 1 of the huntingtin (HTT) gene. Wild-type HTT protein interacts with other proteins to protect cells against toxic stimuli, mediate vesicle transport and endocytosis, and modulate synaptic activity. Mutant HTT protein disrupts autophagy, vesicle transport, neurotransmitter signaling, and mitochondrial function. Although many of the activities of wild-type HTT protein and the toxicities of mutant HTT protein are characterized, less is known about the activities of HTT mRNA. Most putative HD therapies aim to target mutant HTT mRNA before it is translated into the protein. Therefore, it is imperative to learn as much as we can about how cells handle both wild-type and mutant HTT mRNA so that effective therapies can be designed. Here, we review the structure of wild-type and mutant HTT mRNA, with emphasis on their alternatively polyadenylated or spliced isoforms. We then consider the abundance of HTT mRNA isoforms in HD and discuss the potential implications of these findings. Evidence in the review should be used to guide future research aimed at developing mRNA-lowering therapies for HD.

Summary The huntingtin gene has two mRNA isoforms that differ in their 3′ UTR length. The relationship of these isoforms with Huntington's disease is not established. We provide evidence that the abundance of huntingtin 3′ UTR isoforms differs between patient and control neural stem cells, fibroblasts, motor cortex, and cerebellum. Huntingtin 3′ UTR isoforms, including a mid-3′ UTR isoform, have different localizations, half-lives, polyA tail lengths, microRNA sites, and RNA-binding protein sites. Isoform shifts in Huntington's disease motor cortex are not limited to huntingtin; 11% of alternatively polyadenylated genes change the abundance of their 3′ UTR isoforms. Altered expression of RNA-binding proteins may be associated with aberrant isoform abundance; knockdown of the RNA-binding protein CNOT6 in control fibroblasts leads to huntingtin isoform differences similar to those in disease fibroblasts. These findings demonstrate that mRNA 3′ UTR isoform changes are a feature of molecular pathology in the Huntington's disease brain.

In vitro production of germline chimeras and avian cloning may utilise the transfer of avian embryos from their original eggshell to a surrogate eggshell for culture during incubation. Such embryo transfer is valuable for avian cloning as the only alternative would be to transfer the cloned avian embryos into the infundibulum of recipient birds. Given the advances in paleogenomics, synthetic biology, and gene editing, a similar approach might be used to generate extinct species, i.e. de-extinction. One objective of the present research was to examine if ratite eggs could be manipulated via windowing and sham injection, similar to that which could allow for avian genome manipulation and subsequent development. The efficiency of interspecific avian embryo transfer using Chicken ( Gallus gallus domesticus) donor eggs and Turkey ( Meleagris gallopavo) recipient eggshells was also investigated. Egg windowing and embryo transfer techniques utilised in the present research were adapted from those found in the scientific literature. Presumed fertile eggs from Rhode Island Red ( n = 40), Silkie ( n = 2), and White Leghorn Chickens ( n = 18), Turkey ( n = 48), Emu ( Dromaius novaehollandiae) ( n = 79), and Ostrich ( Struthio camelus) ( n = 89) were used in this research. Of the 41 Chicken eggs used for transfers into recipient Turkey eggshells, only one (2.4%) produced a chick. Of 31 windowed Emu eggs, one embryo survived for 25 d but no chicks were produced. Of 36 windowed Ostrich eggs, one embryo survived and hatched. The efficiency of the windowing and embryo transfers to produce chicks was low and further refinements are needed. Importantly, the results herein establish that manipulating ratite embryos is possible.

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.

The objective of the present study was to measure serum levels of obestatin by ELISA with the purpose of establishing parameters and being able to use it as an endocrine marker for the selection of reproductive characteristics of milk and beef cattle. In test 1, 15 Creole cows with genetic tendencies of Charolais, Angus, Brangus, and Gelbvieh were sampled in diestrus and 15 in proestrus, determining their structures with an Aloka Prosound ultrasound with a 7.5-MHz linear transducer. In test 2, 16 Holstein cows were sampled in 2 different ovarian stages, diestrus and proestrus. The standardized technique was a bovine obestatin 96-well ELISA kit from My Biosource Inc. The data were analysed by measuring central tendency and linear regression, where the average levels of obestatin were related to the corpus luteum size and size and number of follicles in milk and beef cattle. We found average levels of 556.99 and 453.35 pg for beef and dairy cattle, respectively (P20mm, 10–20mm, and

Colour-Doppler ultrasonography (CDU) has been applied for a more detailed examination of the ovary and uterus, mainly local blood flow in ovarian follicles and the corpus luteum (CL). The main use of CDU is for the diagnosis of early pregnancy or the selection of recipients for embryo transfer in cattle; however, this tool can also be used to select oocyte donors according to blood flow to the ovary. The concept is that more blood flow in the ovary is conducive to a healthier intraovarian environment (e.g. higher progesterone levels and removal of reactive oxygen species), yielding higher quality oocytes. The objective of this research was to evaluate invitro embryo production (IVP) after selection of oocyte donors, considering ovary irrigation before ovum pickup (OPU). The research was carried out in the reproduction laboratory at the Palominos Ranch (Jalisco, México). The oocyte donors (n=15) were synchronized before each round of OPU using an intravaginal device (1.9g of progesterone) with oestradiol benzoate (2mg) and cloprostenol sodium (500µg) on Day 0, to avoid the presence of a corpus luteum and to synchronize the follicular wave. On Day 6, the intravaginal device was removed and OPU was performed. All Angus breed donors between 3 and 5 years old with a body condition score between 5–6 (scale 1–9, where 1 is extremely thin and 9 is very obese) and were evaluated with transrectal CDU (Sonoscape S2™) with a linear-array probe (7.5MHz) before the first OPU session only. The oocyte donors were classified subjectively into three categories: low blood flow (LBF, ∼30% ovarian area), median blood flow (MBF, ∼50% ovarian area), and high blood flow (HBF, ∼70% ovarian area) and were submitted to 3 cycles of IVP each (45 total cycles). Semen from a proven bull for IVF was used during all IVP cycles. All oocytes collected from each donor were used in IVF with the same semen in all IVP cycles. The total oocytes collected, and percentages of viable oocytes, cleavage, and blastocysts on Day 7 of culture were evaluated. Statistical analysis was carried out using the GLM procedure of SAS software (version 9.3; SAS Institute Inc.) to evaluate the results of LBF, MBF, and HBF (P-value=0.05). Total oocyte recovery was 11.28±1.92, 10.06±1.31, and 15.52±1.05 for HBF, MBF, and LBF, respectively, being significantly higher for LBF (P

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

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.

There is little information in the scientific literature concerning sheep pregnancy and lambing success with regard to the timeframe from when in vitro produced embryos are transported to the designated location for embryo transfer (ET). The aim of this study was to transfer in vitro produced embryos under two different conditions that could typically occur using the aforementioned assisted reproductive techniques (ARTs). Abattoir ovaries were used to procure oocytes for in vitro embryo production and subsequent transfer to synchronized ewes. The study consisted of two experiments: Experiment 1 (Exp1)—embryos taken from the laboratory to a nearby surgical room for immediate ET, and Experiment 2 (Exp2)—ET after 5 hours (h) of transport to a rural farm. Lambing in relation to detected pregnancies, births compared to pregnancies, and the proportion of twin offspring were all higher in Exp2. Notably, in both Exp1 and Exp2, there was not a significant difference (P > 0.05) between the number of embryos transferred, i.e., 3 versus 4, respectively, and the number of ewes that underwent parturition in each group. Also, in both experiments there was not a significant difference (P > 0.05) in the number of ewes that underwent parturition based on the number (i.e., ≥1) of corpora lutea present. The results of the present study demonstrate the importance of evaluating different conditions when applying ARTs, as there are many variables that can influence the outcome. Importantly, Exp2 results show that ovine ET in places located far away from the embryo production site can be useful and successful provided that embryo transport, ET, and recipient conditions are adequate.

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.

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 use of reproductive technologies such as somatic cell nuclear transfer (SCNT) for avian species has been limited by the inability to visualise the pronucleus or pronuclei within the blastodisc or germinal disc region, respectively, primarily due to the opacity of the large, lipid filled yolk. The main objective in the present study was to assess a method for visualising and enucleating the avian ovum, a critical step in developing the capability for cloning birds. The method utilised in the present investigation was epi-fluorescence transmitted light (top-side UV) microscopy (EFTLM), also known as fluorescence/oblique or fluorescence/differential interference contrast (DIC) illumination, combined with vital staining and DNA visualisation techniques. The use of EFTLM combined with micromanipulation methods adapted from mammalian cloning procedures showed that the vitelline membrane of the avian ovum can be pierced and aspiration of the pronucleus, once visualised, can be performed without compromising the ovum's structure. Two approaches for domestic chicken ova collection, i.e., in vivo and in vitro, were utilised and ova recovery rates were compared. Based on a statistical analysis, i.e., Fisher's exact test, the results of the in vivo versus in vitro ovulation ova recovery methods were significantly different (P < 0.05), with the in vivo ovulation method yielding more viable intact ova. In conclusion, enucleating the avian ovum using EFTLM combined with vital staining, DNA visualisation, and micromanipulation techniques can be a feasible option for future avian cloning endeavours; although it will require further refinement to improve overall efficiency.

The aim of this study was to quantify the content of lipid droplets in bovine oocytes and embryos from Bos indicus (Bi), Bos taurus (Bt) and Bos indicus × Bos taurus (Bi × Bt). Oocytes were aspirated post-mortem and subjected to in vitro maturation, in vitro fertilization and in vitro development; the medium employed at each stage (TCM-199, TALP, SOF) was supplemented with (i) serum replacement (SR), (ii) foetal calf serum (FCS) or (iii) oestrous cow serum (ECS). The structure and distribution of the lipid droplets were established using electron microscopy, but were quantified using an optical microscope on semi-fine toluidine blue-stained sections. The highest percentage of embryos corresponded to those produced with FCS and ECS, which differed from embryos generated with SR (p < 0.05). The highest percentage of morulae and the lowest percentage of blastocysts were obtained with the SR supplement (p < 0.05). The oocytes cultured in FCS demonstrated a higher number of lipid droplets compared to those cultured in SR and ECS (p < 0.05). Less accumulation of lipids was observed in embryos supplemented with SR. The lowest and highest numbers of lipid droplets in oocytes corresponded to the Bi and Bt strain, respectively. The lowest amount of lipid droplets in embryos was observed in Bi (p < 0.05). In conclusion, supplementation of the in vitro development culture medium (synthetic oviduct fluid) with a synthetic substitute serum produced similar results in terms of embryo development compared to those obtained with FCS, but a decreased degree of lipid droplet accumulation was observed in the in vitro-cultured embryos.

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 technologies (ART) continue to develop rapidly, allowing for the development of techniques to increase reproductive efficiency and contribute to the genetic improvement of cattle. Some of these techniques include in vitro production (IVP) of embryos and embryo transfer. These modern ART can help produce offspring with highly desirable characteristics. However, there is a lack of information on the percentage of pregnancies obtained following transfer (P/ET) of IVP embryos derived using semen of cloned Bos indicus bulls. The objective of this study was to compare embryo transfer results of IVP embryos created using frozen-thawed semen from 5 Brahman bulls (Bos indicus) with characteristics and genetics of high commercial value. The embryos were produced on two different dates, 45 days apart, using pooled oocytes harvested by ovum pickup from 15 Brahman cows at random stages of the oestrous cycle. Procedures for IVP were performed in a commercial laboratory (Genemex Internacional) in the state of Chiapas, Mexico. For IVF, conventional semen was used from 1 bull (B1) and his clone (B12), the grandson of B1 (B2), and from 2 nonrelated bulls (B3 and B4). A total of 100 embryos were transferred nonsurgically by a private practitioner on a ranch in the state of Campeche, Mexico. The recipients were commercial crossbred cows synchronized using a FTET program. On Day 0, recipients received an intravaginal device containing 1.9g of progesterone (CIDR) and 2mg of oestradiol benzoate IM. On Day 8, the CIDR was removed and cows received 25mg of dinoprost tromethamine, 200IU of eCG, and 0.5mg of oestradiol cypionate IM. Embryos were transferred on Day 17. The overall P/ET was 42% (42/100). The P/ET for IVP embryos produced with semen from bulls B1, B12, B2, B3, and B4 was 3/15 (20%), 3/8 (37%), 23/42 (55%), 8/20 (40%), and 5/15 (33%), respectively. The P/ET was numerically greater for embryos produced using semen from the cloned bull (37%; B12) compared to embryos produced using semen from the original noncloned bull (20%; B1), although this difference was not statistically significant (P=0.62, Fisher’s exact test). There was a significant difference (P

The scientific literature lacks information regarding the incubation conditions and hatching success of the resplendent quetzal (Pharomachrus mocinno mocinno). Jesus Estudillo Lopez, founder of the El Nido aviary, was the first to succeed with quetzal reproduction in captivity. Quetzals do not typically reproduce in captivity and the aim of this study was to increase fecundity through artificial incubation. Over a 3-year period (Yr1, Yr2, and Yr3), data about quetzal eggs, artificial incubation, and diet were recorded for 3 quetzal pairs (ranging from ~4-12 years old, with females ~4-8 years old). Eggs were manually collected from artificial nests and egg substitution was utilised. Quetzals can continue laying eggs up to 8 times with egg substitution. Eggs were weighed and placed into an 80 W incubator (INCA 200®, DMP Engineering, Tel Aviv, Israel) with an automatic egg turner. The eggs were incubated for 17 to 18 days at 37.2 to 37.6°C with a relative humidity of 20 to 40%. Fisher’s exact test (2-tailed, α = 0.05) was used for statistical analyses comparing the numbers of eggs hatched, infertile eggs, and eggs experiencing embryonic death for the study period. Eggs were candled on Day 3 to determine fertility by the presence of a darker area with vasculogenesis. Hatchability (%) was determined by dividing the total number of offspring hatched by the total number of eggs deemed fertile after candling. All of the eggs that did not hatch were opened at the end of the incubation period to detect early embryo death. A total of 27 quetzal eggs [mean weight (g) = 17.3, s.d. = 1.2] were incubated over 3 years, of which 10 (~37%) hatched, with 3 (30%) fledging and surviving to adulthood. Six of the eggs (~22%) suffered embryonic mortality during incubation, whereas 11 eggs were infertile (~41%). Hatchability over the study period was 62.5% (10/16). During Yr1 and Yr2 two eggs hatched each year, and compared with the 6 eggs that hatched in Yr3, a noteworthy but not significant difference was observed (Yr1 v. Yr2, P = 1; Yr1 v. Yr3, P = 0.188; Yr2 v. Yr3, P = 0.170). Embryonic death of the eggs was also not significantly different between the years (Yr1 v. Yr2, P = 1; Yr1 v. Yr3, P = 0.314; Yr2 v. Yr3, P = 0.303). However, the number of infertile eggs was significantly different between Yr1 v. Yr3 (P = 0.007) and Yr2 v. Yr3 (P = 0.003), but not Yr1 v. Yr2 (P = 1). During Yr1 and Yr2 the diet consisted of fruit (aguacatillo, banana, and papaya) and a mouse pup once a week. In Yr3 the diet was modified to include blueberry and a commercial food for Tucans (Mazuri®; https://www.mazuri.com/) and a new quetzal pair was added. The modified diet will continue to be used given that there were fewer infertile eggs. However, future research will need to be conducted to elucidate which variables specifically contribute to better hatching results. The results of this study demonstrate an assisted reproductive technique for quetzals that can benefit conservation efforts for this species.

Multiple ovulation and embryo transfer (MOET) is a biotechnology widely used for genetic improvement in cattle; however, there is great variability in donor response. Differences between management, genotype, environmental conditions, among other factors, can affect MOET results. The traditional protocols consist of 8 twice-daily FSH applications in decreasing dosages over 4 days. Reducing donor handling during MOET is an alternative for embryo production. The aim of this research was to evaluate a simplified protocol, testing single daily administrations of FSH over 4 days, without modifying the total dose calculated for treatment. The study was conducted in northern Mexico. Beef cattle donors (n = 30) were used, 15 with control treatment (CT) and 15 with simplified treatment (ST). All donors were examined beforehand, focusing on the reproductive tract and body score condition, i.e. 5 to 7 (scale of 1-9). Intravaginal devices (IVD) Dispocel Max (Von Franken, Buenos Aires, Argentina) with 1.2 g of progesterone were inserted and 2.5 mg of oestradiol benzoate (Internacional Prode, Mexico) IM was applied on Day 0; FSH (Folltropin-V, Vetoquinol, Lure, France), applications were started on day 4. For CT, the FSH administration was done twice daily (at a 12-h interval), in 8 decreasing doses, and 150 µg of D-cloprostenol (D-CLO; Prostagenol-D, International Prode, Guadalajara, Mexico) was administered IM in the morning and afternoon of Day 6. In the ST, donors were treated with once-daily injections of decreasing doses of FSH over 4 days, and 150 µg of D-CLO was administered IM on Days 6 and 7. The IVD were removed on Day 7 and oestrus was detected using heat detection patches. Artificial insemination was performed using conventional frozen semen at 12, 20, and, in case of persistent heat, 30 h after oestrus detection. Embryo collection was performed 7 days after insemination. The ovarian response was assessed by counting the corpora lutea (CL) detected by ultrasound. Embryo collection was performed by closed method. Analysis of variance was used with the GLM procedure in SAS software (version 9.3; SAS Institute Inc., Cary, NC, USA), to evaluate the results of ST compared with CT (α-level = 0.05). The cattle breed and ranch identification were considered in the analyses. The number of CL in ST and CT was 6.8 ± 1.6 and 8.8 ± 1.5, respectively. The number of oocytes/embryos recovered in ST and CT were 7.3 ± 3.0 and 11.4 ± 2.7, respectively. The number of viable embryos (transferred or frozen) in ST and CT were 2.6 ± 1.7 and 4.7 ± 1.6, respectively. The number of unfertilized oocytes were 2.8 ± 1.5 in ST and 4.2 ± 1.3 in CT. The number of degenerated embryos in the ST were 2.0 ± 1.1 and 1.0 ± 1.0 in the CT. There were no significant differences between groups (P > 0.05) for all variables analysed. Although the number of transferable embryos was numerically higher in the CT group, differences were not significant. Nevertheless, ST can be an alternative superovulation protocol, because working once a day saves time and facilitates the implementation of the MOET technique.

To help save the Sumatran rhino from extinction, the captive breeding program must capitalize on each rhino's reproductive lifespan. Doing so requires knowing when calves are sexually mature. The goal of this study was to monitor physiological changes associated with sexual maturation in two captive born calves (one male and one female) to determine the approximate age of maturity for both sexes of this species. Fecal testosterone metabolites were monitored in the male calf from 6 months to 7 years of age, and fecal pregnane metabolites were measured in the female calf from 6 months to 5.5 years of age. In addition, rectal ultrasonography was employed to monitor changes in ovarian activity from 2 to 5.5 years of age. The male calf's fecal testosterone concentrations reached levels comparable to those detected in samples from adult males when he was 6–6.5 years of age. The first pre-ovulatory sized follicle was observed on the ovaries of the female calf when she was 4.75 years old, but fecal pregnane metabolite concentrations only reached maximum mean concentrations and variability when she was 5–5.5 years of age. Results from this study indicate that male and female Sumatran rhino calves are sexually mature at 6–6.5 and 5–5.5 years of age, respectively. Zoo Biol. 32:549–555, 2013. © 2013 Wiley Periodicals Inc.

The objective was to determine if lipid segregation, with or without post-thaw laser assisted hatching (LAH) of in vitro produced (IVP) bovine embryos, would enhance in vitro survivability and development 24 h post-thaw. On Day 6 of culture (Day 0 = IVF), in vitro produced bovine embryos were divided into three developmental stages: 32-cell (n = 78), compact morula (CM n = 223), and blastocyst (n =56). Embryos within each stage were allocated to the following treatments prior to cryopreservation in 1.5M ethylene glycol: no treatment (Control), 7.5 μg/mL Cytochalasin B for 20 min (CB), or CB with centrifugation (16,000 × g) for 20 min (CBCF). All CB treatments were extended to include embryo freezing. Immediately post-thaw, one-half of the CBCF and Control groups were subjected to zona pellucida drilling (LAH), using the XY Clone® system, creating groups CBCFLAH and LAH, respectively. All thawed embryos were cultured for 24 h and evaluated. No treatment differences were observed for either post-thaw survival or 24 h development. Within the CM stage, CBCFLAH and LAH exhibited a greater number of both total and live cells than Control (total: 69.4, 69.3, 53.0, live: 56.4, 54.7, 39.3 respectively; P < 0.05). In conclusion, LAH post-thaw alone or in combination with CBCF improved embryo viability following cryopreservation.

Inteli-Straw (I-S) devices equipped with radiofrequency identification (RFID) technology were developed for gamete and embryo packaging, storage, and information retrieval to benefit the assisted reproduction industry. The aim of this study was to develop and test software for use with I-S technology. Two types of I-S were used, those with 125 and 134 kHz RFID chips, in conjunction with corresponding wireless RFID readers. Two different RFID chip designs were tested: (1) Mini: 0.25-mL straw, RFID tag dimensions = 1.25 × 7 mm and 1.4 × 8 mm; Standard: 0.5-mL straw, RFID tag dimensions = 2 × 12 mm; and (2) µ-chip (Hitachi Ltd., Tokyo, Japan) of 0.4 mm square and 0.06 mm thick. Inteli-Straw RFID chips can be written or have codes that can be associated with information in a database. The present RFID chips were used with the idChamp DX1 Veterinary & Livestock RFID Reader that uses Bluetooth to connect to an iOS platform for cloud computing. The iScanBrowser (Serialio, TX, USA) LED, an app for an iOS platform for use with a wireless RFID reader, was utilised, as well as a proprietary gamete and embryo database software. The present system allows for quickly accessing the RFID codes from the various I-S gamete and embryo packaging (before or after I-S filling) and entering the data into a computer or cloud-based database that can track their status, movement, metadata, and so on. The ability to acquire I-S information with wireless entry into the cloud-based database was achieved. When the RFID wireless reader detects an I-S, the RFID code is displayed almost immediately (~1 s) in a cell in the software system. The I-S (n = 194) were scanned for automatic database entry. For the wireless-enabled proof of concept, 17 scans of I-S [i.e. RFID chip design 1 (n = 15) and design 2 (n = 2)] were made with wireless scanner detection (< 2.5 cm from RFID reader to I-S) and online database entry with a 100% success rate. By using this I-S method, the present invention provides a wireless, cloud-based system for local or remote access, potentially benefiting both laboratory and field logistics. For AI and embryo transfer, the user can scan an I-S and the information is automatically detected; wireless cloud computing and RFID data crosschecking can occur; and the information can be uploaded to a database for later retrieval or analysis. Further, one can use an iphone or ipad to enter other information such as cow number into the cloud-based database during AI or embryo transfer. The present system can allow for near real-time viewing of the data, locally and remotely, or cross-checking of materials and associated information to reduce errors and improve assisted reproductive technology efficiency.

Although considered a species of least concern by the International Union for Conservation of Nature red list, the Desert Bighorn sheep (Ovis canadensis nelsoni) is listed in Appendix II of CITES (Convention on International Trade in Endangered Species of Wild Fauna and Flora) in Mexico, due to population size and the lack of protected areas. Postmortem epididymal sperm collected from a hunter-harvested Desert Bighorn sheep ram in Mexico, with an unofficial Safari Club International score of 197 2/8 and an estimated 5.5 years old, were used to evaluate the in vitro production (IVP) of embryos using postmortem-collected ram sperm. Testicles with epididymides were placed in the refrigerator ~45 min after harvest. Sperm were extracted from each epididymis and assessed separately for total motility (TM), progressive motility (PM), and membrane integrity using a phase contrast microscope. The sperm suspension was obtained from the distal end of both epididymides and cryopreserved 12 h postmortem using triladyl with egg yolk. Membrane integrity and morphology were evaluated using Eosin-Nigrosin stain. Sperm DNA fragmentation was analysed using the Halomax kit (Halosperm SL, Madrid, Spain) with fluorescence microscopy. Centrifugation with density gradient PureSperm (Nidacon International, Mölndal, Sweden) was used to remove dead sperm and debris before IVF. Ovaries were collected from Domestic sheep (Ovis aries) at a local slaughterhouse. The maturation medium was TCM-199 with Earle’s salts and a modified Tris-buffered medium was used for fertilization. Frozen straws of sperm from the Desert Bighorn ram were thawed for 45 s at 37°C. Sperm were diluted with modified Tween medium B with milk powder (mTBM) to a final concentration of 5 × 106 cells mL−1. The gametes were co-incubated for 18 h under previously described conditions. The cumulus cells were mechanically removed from zygotes and grown using a co-culture with granulosa cells in sequential media SOF1-SOF2. With regard to sperm collection, epididymis 1 produced 29 straws of sperm (0.25 mL, 136 × 106 sperm mL−1) and epididymis 2 produced 32 straws of sperm (0.25 mL, 68 × 106 sperm mL−1). The sperm sample used for IVF had TM of 60% and PM of 30%. Live dead staining of fresh sperm showed 68% live (i.e. intact cell membranes) and 28% post-thaw. Regarding DNA integrity, only 2% of sperm had DNA fragmentation at 0 h. Of 15 Grade 1 oocytes used for IVF, 4 cleaved (27%), with 1 developing to blastocyst stage (25%). The results show that frozen–thawed epididymal sperm collected from a recently deceased Desert Bighorn ram can provide a valuable source of sperm for IVP of embryos. These results also provide new information on Desert Bighorn sheep reproductive parameters for use in health assessment, or reproduction and conservation management through gene banking and assisted reproductive techniques.

The present work reports on a study of plant regeneration carried out with callus from the leaf blades and petioles of field-grown male adult kiwifruit plants (Actinidia deliciosa (Chev.) Liang and Ferguson). The cultivars used were ‘Tomuri’ and clone A, a selected male plant grown in north western Spain. The best shoot induction conditions were obtained in ‘Tomuri’ leaf blades cultured in K(h) medium in the presence of 23 µM Zeatin and 0.1 µM NAA. Under these conditions, more than 80% of organogenic callus induction was observed, with an average of 14 new shoots in the second subculture. The initial length of the shoots affected shoot elongation, which was accomplished by culturing isolated shoots in K(h) medium with half-strength salts, supplemented with 0.4 µM Zeatin and 0.1 µM NAA. A possible detrimental long-term effect of cytokinins on shoot elongation can account for the results, since elongation was not observed until 1 month of culture in elongation medium. For rooting, shoots (1 cm in length) were basally immersed in a 5 mM IBA solution for 15 s, and transferred to half-strength K(h) basal medium. Regenerated plants were acclimated in a sterile peat:perlite substrate for 10 days, and then transferred to soil. AFLP analysis was accomplished with 15 primer combinations from which 13 showed reproducible and well-resolved bands, producing a total of 1321 fragments from which 1281 were polymorphic (97%). A dendrogram was constructed using both monomorphic and polymorphic bands, showing genetic variation among field-grown plants and tissue culture-derived regenerants.

We investigated the characterization of genotypes of Actinidia deliciosa (Chev.) Liang and Ferguson var. deliciosa by using isozymatic and molecular techniques (randomly ampli ed polymorphic DNA (RAPD), ampli ed fragment-length polymorphism (AFLP), standard AFLP, and modi ed AFLP). Four genotypes were tested, the female cultivar 鄂Hayward 鈂, the traditional New Zealand pollinizers 鄂Matua 鈂 and 鄂Tomuri 鈂, and a new pol- linizer named clone A selected in a breeding program in Spain. PGI and PGM were the only isozymes that allowed us to distinguish the kiwifruit genotypes, although the accessions of 鄂Matua 鈂 presented two different banding patterns for both isozymes. All three molecular markers differentiated between the genotypes of kiwifruit tested, although RAPD markers did not allow us to establish differences between accessions of 鄂Matua 鈂, while both standard and modi ed AFLP did. These results, along with those of isozymes, support the hypothesis that the male kiwifruit genotypes present in Europe belong to different clones. None of the markers used showed differences between accessions of 鄂Hayward 鈂, which would suggest that it is a uniform cultivar. On the other hand, clone A was a seedling derived from 鄂Hayward 鈂 and an unknown pollinizer. The results obtained using AFLP markers strongly suggest that 鄂Tomuri 鈂 may have been the male parent of clone A. A speci c protocol for kiwifruit characterization based on a modi ed AFLP technique is also presented, that gave rise to the highest percentage of polymorphism while scoring the lowest number of bands. This, together with the technical features of modi ed AFLP markers, make them very useful for identifying propagated kiwifruit plant material in commercial nurseries. Kiwifruit (Actinidia deliciosa (Chev.) Liang and Ferguson) is a dioecious species which has been commercially planted in Europe since the early 1970s using plants from New Zealand.

A simple and reliable protocol for micropropagation during 12 subcultures of two field growth male plants of kiwifruit [Actinidia deliciosa (A.Chev.) Liang and Ferguson] is described. The best results of shoot multiplication and elongation were obtained in Cheng's K(h) medium in the presence of 0.5 μm NAA, 22 μm BA and 1.4 μm GA3 for `Tomuri' explants, and of 0.1 μm NAA, 4.4 μm BA, and 0.3 μm GA3 for clone A explants. In addition, the cytokinin compounds TDZ and mT were also tested allowing improving the multiplication rate in `Tomuri' explants. For rooting, `Tomuri' and clone A developed shoots were treated by basal immersion in a 5 mm IBA solution for 15 seconds. Treated shoots were then cultured in half-strength K(h) medium without growth regulators showing 100% rooting after 30 days. Regenerated plantlets were successfully transplanted to soil (90% survival) and they are actively growing in the field. Somaclonal variation analysis by AFLP was carried out using 15 primer combinations, yielding reproducible and well-resolved bands with a 57% of polymorphism. AFLP markers showed to be effective to discriminate genetic variation in this species, being greater in clone A than `Tomuri'. Chemical names used: N6-benzyladenine (BA); gibberellic acid (GA3); indole-3-butyric acid (IBA); meta-topolin (mT); naphthaleneacetic acid (NAA); thidiazuron (TDZ).

A study on algae and bacteria population changes, as a function of time, was carried out in a pilot scale oxidation channel bioreactor using a carrousel system. Total Coliforms, Pseudomonas aeruginosa, and Streptococcus faecalis, the most common bacteria found in sewage, Scenedesmus obliquus and Chlorella vulgaris were the microalgae considered in this work. Physicochemical parameters such as COD, BOD, Chlorophyll a, nitrogen, and phosphorous compounds were studied and determined during the experiments. It was demonstrated that the level of wastewater contamination could be predicted based on the bacterial and algae composition. The relationships between the algae and bacteria population, and the variation of these microorganism populations as a measurement of the level of purification were established. The oxidation channel was able to remove a considerable amount of organic matter and pathogenic microorganisms in a relatively short time. The nitrification process could not be measured. The increase in the relative concentration of microalgae contributed toward improving the global efficiency of the system as well as reducing the pathogenic bacteria population.

The actual challenge for the majority of research centers involves the embryo culture medium, since it is known that the culture medium plays a large role in determining embryo quality. Pterostilbene (PT) is a natural analogue of Resveratrol, an antioxidant that can reduce lipids in embryos, but no reports exist of PT being used with IVF-produced embryos or gametes. The objective of the present research was to evaluate the effect of PT in culture media CDM1 and CDM2 on embryo production, cell count, lipid accumulation, and reactive oxygen species (ROS). A total of 4 concentrations of PT and a control were evaluated, i.e. 3, 1, 0.33, 0.11, and 0 μM, in 2 separate experiments. The first experiment was performed using 6 replicates (n = 204) to evaluate blastocyst production (n = 201) and determine the percentage of lipids using the stain Sudan-Black, (n = 100). Hoechst 33258 and propidium iodide were used for determining cell counts. The second experiment was performed using 7 replicates, the effect of using PT (0.33 μM) was compared with a control with 2 O2 concentrations (5 and 20%) for evaluating ROS production (n = 124). Blastocysts without zona pellucida were incubated 48 h at 38.5°C in PBS (without polyvinyl alcohol) with 60 μL of ro-green fluorescent protein. After incubation, 25 μL of 4′,6-diamidino-2-phenylindole (1 mg mL−1) was added and incubated for 5 min. A fluorescence microscope was used and positive ROS particles digitized using Photoshop CS6 and quantified using the program ImageJ®. The data were transformed to arcsin values for subsequent analysis. In the first experiment, ANOVA and least significant difference tests were used to determine statistical significance (Statgraphics). No significant differences were found among treatments (P > 0.05) for the internal cell mass: 3, 1, 0.33, 0.11 μM PT (29.2 ± 5.2; 28.9 ± 3.8; 22.2 ± 3.2; 29.0 ± 1.7, respectively) and the control (27.0 ± 1.7). The cells of the trophectoderm did not differ (P > 0.05) between treatments (31.7 ± 4.8; 31.3 ± 3.9; 38.6 ± 3.5; 30.8 ± 1.8) and control (33.4 ± 2.1). Total cells did not differ (P > 0.05) between treatments (72.0 ± 9.8; 82.6 ± 4.3; 94.8 ± 12.8; 73.2 ± 9.2) and control (83.8 ± 7.7). Embryo production (Day 8) was greater for control (33.5 ± 3.0) versus treatments (14.1 ± 1.7; 19.4 ± 1.9; 21.1 ± 2.6; 20.8 ± 2.1) (P 0.05); however, the PT had a significant effect on the reduction of ROS (P

The assisted reproduction industry involving sales and services for gametes and embryos for domestic animals of commercial value is a large market totaling millions of dollars annually. The objective of this study was to develop and test gamete and embryo packaging—Inteli-Straws (I-S) equipped with radio-frequency identification (RFID) technology. Specifically, French straws (0.25 and 0.5 mL) were modified to include extreme cold-tolerant RFID microchips. Two groups of I-S were formed: Group (G)1: RFID chips that were autoclaved (n = 49), and G2: RFID chips that were not autoclaved (n = 47). Both groups had a control that was not exposed to liquid nitrogen (LN). Each group was exposed to LN up to 4 times: 2 slow freezes first and then 2 fast (i.e. vitrification) freezes, and I-S RFID chip survival was determined. I-S detection and readability (non-autoclaved) was also measured, placing I-S just above LN (in vapors, n = 43) or just below LN (n = 38). Statistical differences (α = 0.05) were determined using Fisher’s exact test. The results between G1 and G2 were not significantly different (P = 0.108) after 4 rounds of cryopreservation (and thawing). For G1, 98% (48/49) of the I-S RFID chips remained operational, and control and treatment were not significantly different (P = 1.000). For G2, 89.4% (42/47) of the autoclaved RFID chips remained operational, and control and treatment not significantly different (P = 0.099). RFID chip readability results; that is, the ability to detect the I-S versus not able to detect the I-S, comparing placement just above liquid nitrogen (LN) versus the placement just below LN were not significantly different (P = 0.105). Notably, detection differences varied within each group, with I-S in G1 (mean = 9.5; SD = 3.5 cm) readable at a larger distance, 5.2 cm farther than the mean of G2 (mean = 4.3; SD = 1.9 cm). During AI or embryo transfer (ET), a technician may not clearly identify the label or colour of straw, may incorrectly record the information, or may take more time than desirable to record it. Increased exposure times may lead to decreased viability of gametes and embryos. The results show that by using the I-S, one may quickly scan the straw within LN or LN vapors, thereby automatically detecting information and even uploading it to a database (e.g. scanner sophistication). We are not aware of comparable device to I-S for locating and retrieving associated information without removing the gamete/embryo packaging from LN or LN vapors; unlike traditionally labelled straws (e.g. laser etched or ink labels). Also, for AI and ET, the I-S can be quickly scanned and the straw information automatically detected and uploaded to a database.

1. The presence of contiguous beds of submerged (Myriophyllum spicatum, Ceratophyllum demersum and Najas marina) and floating-leaved (Trapa natans) vegetation in a north Italian lake allowed us to test the effect of the different host architecture on epiphytic algae and invertebrates and to predict the consequences for the lake of changes in the predominant vegetation. 2. Epiphyton development, measured as carbon, nitrogen, phosphorus, chlorophyll a (Chl a), phaeophytin and as algal and macroinvertebrate density, was significantly higher on submerged plants than on T. natans. The C : Chl a ratio, a proxy of the ratio of heterotrophs to autotrophs, was higher on the floating-leaved plants. The elemental (C : N : P) and pigment (Chl a : phaeophytin) ratios were not significantly different between the two vegetation types. 3. The taxonomic composition of epiphytic algae and invertebrates was similar on the different plants. The more varied morphology of the floating-leaved T. natans resulted in a higher diversity of epiphytic algae, however, but not of macroinvertebrates. 4. There was a significant inverse relationship between epiphyton biomass and the standing crop of the host plant, suggesting a key role for light and water exchange in epiphyton development. 5. Replacement of floating-leaved by submerged plants would increase the total biomass of epiphytic algae and invertebrates.

Diversity of phytoplankton assemblages from the shallow hypertrophic lake, the Albufera of Valencia (Spain), was studied for the period 1980 - 1988. The lake serves as a reservoir for the surrounding rice cultivation, with two seasonal periods of water retention to keep ricefields flooded. The seasonal hydrological cycle seems to be the primary factor driving phytoplankton community changes, compounding the disturbances due to water mixing. The lake is dominated throughout by filamentous cyanophytes. A Shannon-Weaver function was used to measure phytoplankton diversity. Phytoplankton steady-states, defined as the periods when more than 80 % of total phytoplankton biomass was shared by only 1-3 species, were assumed for comparative purposes. Phytoplankton diversity and the assemblages showed a high degree of interannual regularity during the study period. Phytoplankton was mainly limited by light and water flushing from November to April, and more by nitrogen and phosphorus during summer. Low diversities were related to stable community states, while higher diversities corresponded to non-steady state periods, the former lasting longer. Four annual phytoplankton associations were found, both steady and non-steady states being characterized by two different algal communities. Diversity reached a steady-state, lasting from November to june. From january to March, water flushing high nutrient levels and oligophotic conditions selected in favour of large unicellular phytoplankton species. The onset of spring quiescence resulted in the lowest values of diversity and species richness, due to the overwhelming dominance of vernal crops of Planktothrix agardhii, whose selfshading populations would have excluded many less well-adapted species. By june, nutrient decrease and increasing temperatures and irradiance, moved phytoplankton towards an intermediate assemblage dominated by smaller and faster growing species. Thereafter, long-summer water stabilization and nutrient depletion interacted to enhance diversity and the onset of a non-steady state, characterized by an immature summer assemblage of large colonies, potentially motile (e.g. Microcystis) and with high nutrient storage capacity. During the autumnal period when the ricefields are drained for harvest, diversities reached maximal levels, including species from the surrounding habitats (ricefields and drainage ditches). Lake reisolation by November-December led to another phytoplankton steady-state, when the phytoplankton reverted toward a composition similar to that of June. Storms increased diversity and the presence of small-sized species, contributing to a more rapid species replacement or arresting the establishment of an steady-state, depending upon their intensity frequency and occurence. Stagnation always selected in favour of cyanophytes, while flushing rates enhanced the presence of other algal groups, mainly diatoms. The annual flushing cycle of the lake is regarded as a potential tool for the future lake restoration. Our results confirm the view of regarding the phytoplankton of polymictic Oscillatoria-dominated lakes, as sub-climatic communities mainly limited by light, and agree with some postulates based on the IDH-diversity theory

The effects of light on the steps of embryo manipulation have been described in several species, including humans. There are reports in which exposure of these cells to UV rays from sunlight affects them epigenetically, which could lead to meiotic arrest that would prevent normal maturation from the germinal vesicle (GV) to metaphase II (MII) stage, which would compromise subsequent embryo viability. Development to the blastocyst was not evaluated. The objective of the study was to observe the difference in maturation capacity (GV to MII) of prepubertal gilt oocytes under conditions of reduced ambient light. Thirty Duroc ovaries were recovered at slaughter and immersed in saline (0.9% NaCl) supplemented with penicillin-G (100 IU mL–1) and streptomycin sulfate (100 mg mL–1) at 29 to 34°C for transport to the laboratory where they were punctured with an 18-gauge needle attached to a 10-mL syringe. GV oocytes were selected with at least 3 layers of compact cumulus cells and were incubated with TCM-199 for 42 h in total with replacement with fresh maturation medium at 22 h. Oocytes were subsequently denuded using 0.1% hyaluronidase, fixed with 2% formaldehyde, and stained with Hoechst 33342 (10 min). Thereafter, oocytes were evaluated under a fluorescence microscope for the stage of meiosis: GV, metaphase I (MI), anaphase I/telophase I (ATI), and MII. GV oocytes were divided into two groups of 60 each as follows: Group 1 = handling in ambient light (1 change of culture medium and evaluation at the stereoscope); Group 2 = handling with reduced light (the change of medium was carried out in a dark room with red light on the stereoscope. Chi-square and Student’s t-test with Minitab 16.0 statistical program were used, and differences were considered significant at P

Given previous low sperm decondensation rates and poor oocyte activation in sheep ICSI (10–20%), we evaluated activation techniques for IVM/ICSI. Incubations were performed in a 5% CO2 cell incubator at 38.5°C and saturated humidity. Sheep ovaries were collected at an abattoir and transported

Biotechnology continues to evolve rapidly, allowing the development of artificial reproductive techniques (ART) to increase reproductive efficiency and contribute to the genetic improvement of domestic animals. The present study examines the results obtained after 30 months of starting a commercial practice for ovum pickup (OPU) in vitro embryo production (IVP) and embryo transfer (ET) in cattle in tropical and subtropical Mexico. This research was conducted from 2013–2015 in beef and dairy cattle kept under different environmental and management conditions in 6 states (Chiapas, Oaxaca, Tabasco, Tamaulipas, Veracruz, and Yucatan). Oocytes were collected by OPU, without hormonal treatment, from 10 donor cows: 2 Bos taurus (Bt), 5 Bos indicus (Bi), and 3 Bt × Bi. A total of 98 oocyte recovery sessions were performed on 756 cows and produced 12 524 viable oocytes (1349 GI, 3383 GII, 7792 GIII), which were sent to a central laboratory for IVP. Both conventional and sexed semen were used for IVF, from 9 breeds: 2 Bt, 5 Bi and 2 Bt × Bi. The overall cleavage rate was 69% (8587/12 524). The embryo production efficiency rate was 31% (3905/12 524). Fresh sexed and conventional embryos were transferred to recipients synchronised with the following protocol: Day 0, application of an intravaginal device (ID) with progesterone and 2 mg of oestradiol benzoate IM; Day 8, removal of the ID, 400 IU eCG IM, 0.5 mg cloprostenol sodium IM, and 0.5 mg oestradiol cipionate IM. Day 10 was considered the day of oestrus. Pregnancy rate after ET was 38% (945/3905). The average number of viable oocytes per donor cow was 16.57; the average number of transferred embryos per donor cow was 5.17, and the average number of pregnancies per donor cow was 1.25. The OPU-IVP were successful in producing pregnancies even under several adverse conditions, such as a tropical environment, many donors being prepuberal females and the majority of the adult cows having a previous non-productive history due to hormonal mishandling in superovulation programs or because of overfeeding for show purposes. The consequences of these factors can be observed in the lower overall cleavage rate obtained (69%), compared to the expected (75%). Some variables require further analysis (e.g. different OPU and ET technicians, time of year, cow age, cow breed, and use of conventional and sexed semen). A successful IVP practice has to face and overcome multiple problems that may arise in some geographic locations but, perhaps, not in others. It is interesting to note the use of sexed versus conventional embryos, of the 98 ET sessions, 25 involved sexed embryos versus 73 for conventional embryos. The use of sexed semen for OPU-IVP and ET in this region of Mexico continues to expand. We thank Genemex Internacional and the ranch owners that were involved with this research.

A decrease in fertility can have a negative economic impact, both locally and over a broader geographical scope, and this is especially the case with regard to the cattle industry. Therefore, much interest exists in evaluating proteins that might be able to increase the fertility of sperm. Heparin binding proteins (HBPs), specifically the fertility associated antigen (FAA) and the Type-2 tissue inhibitor of metalloproteinase (TIMP-2), act to favor the capacitation and acrosome reaction and perhaps even modulate the immune system’s response toward the sperm. The objective of this research was to determine the effect on fertility of adding recombinant FAA (rFAA) and recombinant TIMP-2 (rTIMP-2) to bovine semen before cryopreservation for use in an artificial insemination (AI) program in a tropical environment. For this experiment, 100 crossbred (Bos taurus x Bos indicus) heifers were selected based on their estrus cycle, body condition score (BCS), of 4 to 6 on a scale of 1 to 9, and adequate anatomical conformation evaluated by pelvic and genital (normal) measurements. Heifers were synchronized using estradiol benzoate (EB), Celosil® (PGF2α) (Shering-Plough) and a controlled internal drug release (CIDR) device was inserted that contained progesterone. Inseminations were performed in two groups at random, 50 animals per group. The control group was inseminated with conventional semen. The treatment group was inseminated with semen containing rFAA (25 µg/mL) and rTIMP-2 (25 µg/mL). In the control group a 16% pregnancy rate was obtained versus a 40% pregnancy rate for the HBP treatment group, resulting in a significant difference (P = 0.0037). Given the results herein, one may conclude that the HBPs can increase fertility and could be an option for cattle in tropical conditions; however, one needs to consider the environment, nutrition, and the genetic interaction affecting the final result in whatever reproductive program that is implemented.