Assisted Reproductive Technologies as Veritable Tools for Improving Production Efficiencies of N’dama and Muturu Cattle Breeds in Nigeria-A Review

Assisted reproductive technologies (ART) that have come to stay and are still being improved upon in developed countries are still in their infancy stage in developing countries like Nigeria. Nigeria’s cattle population is estimated to be around 18.4 million. The number is far insufficient to meet the country’s demand for meat, milk, and other cow products, let alone contribute to GDP. N’dama and Muturu are both Nigerian breeds that are resistant to trypanosomosis. They are humpless longhorn and humpless shorthorn types of beef cattle. The dairy and beef cow industries’ inadequate adoption of ART is partly to blame for Nigeria’s low cattle output. Sex determination, multiple-ovulation and embryo transfer (MOET), oestrus synchronization, artificial insemination (AI), in vitro fertilization (IVF), cloning, and genetic engineering are all examples of assisted reproductive technologies. It has been reported in humans, rodents and domestic animals, abnormal fetuses, newborns and adult offspring arise from ART. Improper matching of breeding animals mostly leads to overfat calves. This review centers on the applications and potentials of ART in the production of trypanotolerant N’dama and Muturu cattle breeds. Some unorthodox medicines which have proven effective in human reproduction can circumvent the shortfalls in the adoption of ART.

Superovulation response of Peranakan Ongole (PO) and Simmental cows after FSH stimulation in multiple ovulation and embryo transfer program

Multiple ovulation and embryo transfer (MOET) is a reproductive technology to increase the livestock population in a short period. The success of ovary stimulation programs is influenced by an individual’s response to follicle-stimulating hormone (FSH) stimulation. In this study embryo production was carried out on local cows represented by Peranakan Ongole (PO) cows and exotic cows represented by Simmental cows. FSH stimulation was performed on 10 PO cows and 10 Simmental cows. On Day-1 Cue Mate® (progesterone) was inserted intravaginally. FSH injection (400 mg) was carried out intramuscularly from D-10 at reduced dose with 12 hours intervals (Day-10: 100 mg; Day-11: 60 mg; Day-12: 40 mg per injection). On Day-12, prostaglandin (PGF2α) was injected and Cue Mate® was removed. Artificial insemination (AI) was performed 48 hours after PGF2α injection for 3 times at 12 hours intervals. The embryo was collected 6 days after the last AI (Day-21). Superovulation response was detected on 70% of PO cows and 90% of Simmental cows. The average number of transferable embryos in Simmental cows (9.11±7.27) was higher than PO cows (7.86±7.78). This research shows that Simmental cows are more responsive to FSH stimulation, and can produce more transferable embryos than PO cows.

Dual extrauterine ectopic pregnancy: double management

A 30-year-old nulliparous woman was referred with suspected left ovarian ectopic pregnancy. She had undergone laparoscopic left salpingectomy for ruptured tubal ectopic pregnancy 3 weeks earlier, following treatment with medications for ovulation induction. Sonological examination revealed a left ovarian ectopic pregnancy corresponding to 8 0/7 weeks with cardiac activity. She underwent ultrasound-guided intrasac therapy with intrasac instillation of 3 mEq of potassium chloride followed by 50 mg of methotrexate. She was followed with weekly measurements of serum beta human Chorionic Gonadotropin (hCG) which returned to baseline after 65 days of the intrasac therapy. This case not only highlights the need for continued follow-up of the serum beta hCG after definitive management of an ectopic pregnancy in cases with multiple ovulations, but also the option of medical management in cases of advanced ovarian ectopic pregnancy. It also accentuates the necessity for adequate counselling to avoid conception in a multiple ovulation cycle.

The Incidence of Ovulation and Detection of Genes Associated with Ovulation and Twinning Rates in Livestock

Cattle is a monotocous species that generally produce only one offspring per conception. However, multiple ovulations are a naturally emerging reproductive phenomenon typically controlled by genetic structure and environmental factors. On the other hand, few genes or causative mutations might explain significant genetic variations between animals for the reproductive traits. Studies report different methods, including QTL analysis, fine mapping, GWAS, and MAS selection, to improve such traits due to their economic importance. The recent fine-mapping study, which narrows the genomic region, indeed, influencing multiple ovulation, gives positive signals that causative mutation controlling high ovulation rate may be identified shortly. In conclusion, identifying the major genes that considerably affect ovulation and twinning rates provides the opportunity to increase reproduction efficiency by improving genetic gain in livestock species.

Early Embryo Exposure to Assisted Reproductive Manipulation Induced Subtle Changes in Liver Epigenetics with No Apparent Negative Health Consequences in Rabbit

Embryo manipulation is a requisite step in assisted reproductive technology (ART). Therefore, it is of great necessity to appraise the safety of ART and investigate the long-term effect, including lipid metabolism, on ART-conceived offspring. Augmenting our ART rabbit model to investigate lipid metabolic outcomes in offspring longitudinally, we detected variations in hepatic DNA methylation ART offspring in the F3 generation for embryonic exposure (multiple ovulation, vitrification and embryo transfer). Through adult liver metabolomics and proteomics, we identified changes mainly related to lipid metabolism (e.g., polyunsaturated fatty acids, steroids, steroid hormone). We also found that DNA methylation analysis was linked to changes in lipid metabolism and apoptosis genes. Nevertheless, these differences did not apparently alter the general health status. Thus, our findings suggest that ART is likely to be a player in embryo epigenetic events related to hepatic homeostasis alteration in adulthood.

Effectiveness of embryo transfer in cows - risk factors including in vivo derived and in vitro produced embryos

Multiple Ovulation Embryo Transfer is a biotech method with more than 50 years of history and an established position in cattle breeding. This procedure is beneficial in many ways, but it also carries a risk of failure. The study presents the overview of the most important risk factors that may affect conception rates in the course of embryo transfer, including the factors associated with the embryo sourcing (embryo production method, embryo quality, development stage and breed, embryo storage method), embryo transfer procedure (synchrony/asynchrony, embryo transfer difficulty, the time of the transcervical insemination gun passage, depth of embryo deposition, localization and structure of the corpus luteum relative to the follicle and both individual characteristics of donors and recipients (level of concentration of progesterone, the state of health of the udder, lactation level, body condition score and age) and some environmental factors.

Ovarian response to P4-PGF-FSH Treatment in Suffolk sheep and P4-PGF-PMSG Synchronization in Cross-bred Ewes, for IVD and ET Protocol

BackgroundThe success of an embryo transfer protocol in sheep depends on many factors, but the choice of drugs for the desired superovulation as well as the conception rate are most essential. Reproductive activity in sheep is characterized by a seasonality influenced by several factors such as photoperiod, latitude, temperature, nutrition and breed. Reproductive seasonality and nutritional condition are the main factors that influence embryo production in sheep. In sheep, some anatomical peculiarities limit the application of traditional reproductive biotechnologies used in cattle. MethodsIn vivo embryo production is often referred to as “multiple ovulation and embryo transfer” and involves ovarian superstimulation of the donor female, insemination or mating, uterine flushing for embryo recovery, and either cryopreservation or transfer of collected embryos to recipients. A total number of 60 sheep and 3 rams were included in this study, divided into 2 groups (receptors/donors). Donor Suffolk sheep were treated for superovulation using the P4‐PGF‐FSH protocol while the cross-bred recipients’ group was synchronized with P4-PGF-PMSG. ResultsOn the first day after superovulation, all ovaries had more than 5 dominant follicles, while corpora lutea were later observed in 83.3% sheep. The recovery rate was 83.3% while 72,9% embryos were transferable. Embryos were transferred directly into recipients. Fertility after 30 days was 68.57%, lambing rate was 91.6%, and CR 62.85%. This study showed that veterinary drugs (P4, FSH, LH, PMSG, PGF) used for superovulation were capable to induce estrus and synchronize ovulation in sheep, are topical and in increasing use worldwide. ConclusionsThe aim of this study was to conclude on the effectiveness of a wider on farm in vivo embryo transfer development program in Suffolk sheep, using several veterinary hormones. The application of a multiple ovulation embryo transfer (MOET) protocol has a positive effect in the production of in vivo derived embryos in Suffolk sheep and can guarantee the success of embryo transfer activity to ewes with lower genetic merit. Our research aimed at representing a model for sheep farms for a rapid improvement of productive traits.

77 Occurrence of early regression of corpora lutea in Dorper ewes subjected to a conventional superovulatory regimen

The early regression of corpora lutea (ERCL) is a functional alteration that occurs more often in animals subjected to multiple ovulation followed by embryo transfer (MOET) technique. Although it is mainly reported in goats, sheep are also susceptible to this disorder. The ERCL may compromise the quality and viability of embryos, and even embryonic recovery rate. Thus, the non-diffusion of the animal genetic material on a commercial scale increases costs, decreasing efficiency. This study aimed to assess the occurrence of ERCL in embryo donor ewes subjected to the MOET programs in different seasons. The research was carried out in a commercial herd in São Luís do Paraitinga city (23°22′S and 45°26′W), Brazil, over 4 years (2017–2020). Forty-four Dorper multiparous ewes aged between 3 and 9 years old and with body condition score (BCS) between 2 and 4.5 (1 to 5 scale) were used. Each ewe was used at least once for MOET and a maximum of 5 times, totalling 104 procedures. Regardless of the day of the oestrous cycle or anovulatory period (Day 0), ewes received a conventional superovulatory protocol consisting of an intravaginal device treatment with 0.33mg of progesterone (CIDR®, Zoetis). On Day 7, the device was replaced with a new one and ewes administered i.m. 0.24mg of sodium cloprostenol (Sincrocio®, Ourofino). The superovulatory treatment [256mg of FSH (Folltropin®, Vetoquinol)] started on Day 12 and consisted of decreasing doses (20, 20, 15, 15, 10, 10, 5, and 5%) administered intramuscularly (IM) every 12h for 4 days plus 200IU of equine chorionic gonadotrophin (eCG, Novormon®, Zoetis) at device removal on Day 14 and 0.1mg of gonadotrophin-releasing hormone (Fertagyl®, MSD) IM 1 day later (Day 15). Laparoscopic AI was performed twice on Day 16 using cooled semen. Five days after AI, ovaries were assessed by laparoscopy to check the presence and viability of corpora lutea (CL). Ewes that had avascular CL (pinkish to whitish colour) were classified as ERCL and embryo collection was not performed. The occurrence of ERCL in each season and category of BCS was checked by either chi-squared or Fisher test. Logistic regression was performed according to the incidence of ERCL in each category of age. Values of P<0.05 were considered as significant. From 104 procedures, ERCL was identified in 26 cases, totalling 25% of occurrence. The proportion of occurrence did not differ (P>0.05) among seasons: breeding (10/43: 23%), transition (10/36, 28%), or anoestrous (6/25, 24%). There was no difference in ERCL incidence in ewes presenting different BCS categories: lower/thin (2 to 2.5: 3/12, 25%); average/good (3 to 3.5: 15/66, 22%) and higher/fat (4 to 4.5: 8/26, 31%). Indeed, there was no association (P>0.05) between ERCL and age. In conclusion, a relevant occurrence of ERCL was detected in superovulated embryo donor ewes but this incidence was not associated with season, age, or BCS of Dorper ewes. These data highlight the importance of pharmacological measures to control ERCL in MOET protocols for commercial sheep herds.

149 Uptake of C18:0 from culture media during invitro culture decreases cryosurvival rates of bovine embryos

Cryosurvival of invitro-produced bovine embryos is lower than that of invivo-produced embryos, limiting their usability in the field. Previous work showed that the embryo’s lipid composition relates to its quality and cryosurvival. The present study aimed to investigate the effects of free fatty acid (FA) additions to embryo culture media during the oviduct phase of embryonic development on the improvement of cryosurvival of invitro-produced blastocysts. Bovine cumulus–oocyte complexes (n=1675, 3 replicates) were harvested from slaughterhouse ovaries, invitro matured (23h), and subsequently fertilized (18–20h). Embryos were cultured until Day 5 post-fertilization in synthetic oviductal fluid (SOF) with (1) bovine serum albumin (BSA; control, n=253); (2) delipidified BSA (>96% FA free, n=460); (3), delipidified BSA complexed with 25µM unsaturated oleic acid (C18:1, n=455); or (4) with saturated stearic acid (C18:0, n=507) with a stoichiometry of 5:1. At Day 5, SOF was refreshed and embryos were cultured without supplementation. At Days 7 and 8, blastocyst rates were determined. Blastocysts were LD540 stained for lipid droplets (LD), and the LD number and size were analysed by ANOVA. Cryosurvival%, defined by re-expansion of the blastocoel, was analysed by logistic regression. Additionally, fresh and frozen–thawed blastocysts were stained for apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labelling, TUNEL), necrosis (EthD-1), and DNA (Hoechst 33342) and analysed using negative binomial regression. Group differences were tested using a post hoc Tukey test. Statistical analysis was performed in R Studio (version 3.4.2), and P-values <0.05 were considered significant. FA-free culture delayed and decreased blastocyst rates to 19% compared with any FA supplementation: 35%, 27%, and 29% for control, C18:1, and C18:0, respectively (P<0.04). Cryosurvival doubled with culture in FA-free SOF (58%) and C18:1 (63%) compared with C18:0 (23% P=0.01 and P<0.01, respectively) and control (29%; P=0.15 and P<0.02, respectively), approaching cryosurvival rates of donated multiple ovulation embryo transfer (MOET) embryos (CRV Company; 67%). C18:0 exposure also resulted in elevated necrosis levels after cryopreservation (5–8% of cells), compared with all groups (2–4%; P<0.016). The LD size increased in blastocysts cultured with C18:1 compared with all groups (3.1µm2 vs. 2.4–2.7µm2; P<0.016). C18:0 addition to SOF during embryo culture invitro, as well as a mixture of FA in control SOF (including C18:0), caused a reduction of ∼50% in blastocyst cryosurvival compared with MOET blastocysts. Interestingly, either C18:1 addition or the complete omission of FA in SOF during embryo culture invitro restored the cryosurvival of blastocysts to the level of MOET blastocysts. Currently, we are investigating whether the free FA conditions in the oviduct endorse the distinct quality between invivo- and invitro-produced embryos.