Production, Evaluation, Cultivation and In vitro Fertilization of Cattle Oocytes: Recent Trends in Reproductive Biotechnologies in Animal Breeding : A Review

The article presents the results of assessing reproductive biotechnology for cattle breeding. The issues of obtaining genetic material from bulls-producers and oocytes from donor cows, for their further cultivation and obtaining embryos, in order to replicate highly productive offspring from valuable animals, are considered. Oocyte production was analyzed in three different ways: puncture, section, and aspiration. A total number of 156 oocyte-cumulus complexes (OCCs) were collected out of 40 ovaries by puncture – 50 pcs, section – 47 pcs, and aspiration – 59 pcs. The results showed that puncture and section gave significantly higher total OCCs per an ovary (4.16 and 4.0, respectively) than aspiration (3.68), but a higher number of normal (grade A and B) OCCs per an ovary was observed with aspiration (2.5) than with puncture (1.82) and section (2.00). During aspiration, oocyte-cumulus complexes were collected from the surface of follicles with a diameter of 3 to 8 mm using a needle. During puncture, all surfaces were pierced with a hypodermic needle; during section, incisions were made along the entire ovarian surface with a scalpel, that is, all sizes of superficial follicles were collected. OCCs were divided into 4 classes based on cumulus and nucleus cells: grade A – oocytes completely surrounded by cumulus cells; Grade B – oocytes partially surrounded by cumulus cells; Grade C – oocytes not surrounded by cumulus cells; and grade D – degeneration observed in both oocyte and cumulus cells. Grades A and B were considered normal, while grades C and D were considered to be damaged. The obtained oocytes were cultured and fertilized in vitro, which means that their fertilization with sperm occurred under artificially maintained optimal conditions outside the body. Normal fertilization results are zygote formation with male and female pronuclei (PN). As a result of the conducted fertilization of 251 mature oocytes, 142 fertilizations were successful, which amounted to 56.57%.

Effects of the donor factors and freezing protocols on the bovine embryonic lipid profile

Embryo lipid profile is affected by in vitro culture conditions, that lead to an increase in lipids. Efforts have been made to optimize embryo lipid composition as it is associated with their quality. The objective of this study was to evaluate whether the diet supplementation of donor cows (n-3 or n-6 PUFA), or the slow freezing protocols (ethylene glycol sucrose EG-S vs. glycerol trehalose GLY-TRE), or the physiological stage of the donor (nulliparous heifers vs. primiparous lactating cows) may impact the bovine embryo lipid profile. Lipid extracts of 97 embryos were individually analysed by liquid chromatography-high resolution mass spectrometry, highlighting 246 lipids including 85% being overabundant in cow embryos compared to heifer embryos. Among 105 differential lipids, 72 were overabundant after EG-S protocol, including a single glycerophosphate PA(32:1) representing 27.3% of the significantly modulated lipids, suggesting that it is degraded when GLY-TRE is used. No lipids were different according to the n-3 or n-6 supplementation of the donor cows. In conclusion, the embryonic lipid profile was mainly affected by the physiological stage of the donors and the slow freezing protocols. The overabundance of lipids in lactating cow embryos and the resulting lower quality of these embryos is consistent with the lower pregnancy rate observed in cows compared to heifers. Unlike GLY-TRE protocol, EG-S freezing allowed to preserve glycerophospholipids potentially improving the slow freezing of in vitro-produced embryos. Further studies are required to modulate embryo quality and freezability by modulating the lipidome and integrating all stages of embryonic production.

Estimation of genetic parameters for superovulatory response traits in Japanese Black cows

The aim of this study was to estimate genetic parameters for superovulatory response traits in order to explore the possibility of genetic improvement in Japanese Black cows. 19 155 records of the total number of embryos and oocytes (TNE) and the number of good embryos (NGE) collected from 1532 donor cows between 2008 and 2018 were analyzed. A two-trait repeatability animal model analysis was performed for both. Because records of TNE and NGE did not follow a normal distribution, the records were analyzed following no, logarithmic, or Anscombe transformation. Without transformation, the heritability estimates were 0.26 for TNE and 0.17 for NGE. With logarithmic transformation, they were 0.22 for TNE and 0.18 for NGE. With Anscombe transformation, they were 0.26 for TNE and 0.18 for NGE. All analyses gave similar genetic correlations between TNE and NGE, ranging from 0.60 to 0.71. Spearman’s rank correlation coefficient between breeding values of cows with more than 10 records was ≥0.95 with both transformations. Thus, the genetic improvement of TNE and NGE of donor cows could be possible in Japanese Black cattle.

GnRH or estradiol benzoate combination with CIDR improves in-vivo embryo production in bovines (Bos indicus and Bos taurus) under subtropics

Multiple Ovulation and Embryo Transfer (MOET) technology is a potential technique to upgrade livestock species’ genetics. The varied response to super-stimulatory treatments remains one of the limiting factors to this technology’s widespread use. The present study was aimed to improve the superovulation response and in-vivo embryo production by using controlled internal drug release (CIDR)-GnRH or CIDR-EB (Estradiol Benzoate) along with conventional superovulation protocol in Holstein Frisian (HF): Bos taurus; n = 42) and Crossbred (XB: Cholistani (Bos indicus) × HF; n = 28) cows. In the CIDR-GnRH/CIDR-EB treatment, CIDR was implanted in the cows after confirming the presence of a corpus luteum (CL) on the 8th day after estrus. 2 ml GnRH (Lecirelin acetate 0.0262 mg/ml) or 2 mg EB was also administered in CIDR-GnRH/CIDR-EB groups, respectively. Both groups were given super-stimulatory treatment from the 11th day after estrus (FSH in tapering doses twice a day for four consecutive days). On day 13, two doses of 2 ml prostaglandin (75 µg/ml of dextrorotatory cloprostenol) were administered (am: pm), and CIDR was removed the following day. Two artificial inseminations (AI) of the cows were performed (12 h apart) on the 15th day. No CIDR and GnRH/E.B were given in the control group, but the remaining superovulation protocol was the same. Later on, seven days after the first AI, non-surgical embryo flushing was done. The transferable embryos produced from three different superovulation protocols were then transferred into the recipient cows (n = 90) for determining their fertility. Statistical analysis revealed that the number of super-estrus follicles (SEF), multiple corpora lutea (MCL), ovulation/fertilization percentage, fertilized structures recovered (FSR), and transferable embryos (TEs) remained significantly higher (p < 0.05), and days taken for return to estrus (RTE) after embryo collection remained significantly lower (p < 0.05) in CIDR-GnRH (n = 18) and CIDR-EB (n = 15) groups as compared to the control (n = 37). The comparison between XB and HF cows revealed that the TEs production in CIDR-GnRH (XB = 5 vs HF = 13) and CIDR-EB (XB = 6 vs HF = 9) based superovulation protocols were 11.60 ± 4.08 vs 04.31 ± 0.98 and 09.33 ± 1.78 vs 05.22 ± 1.36, respectively. TEs production in XB cows (n = 5) of the CIDR-GnRH group was significantly higher (11.60 ± 4.08) than other groups. On the other hand, the days taken for RTE after embryo collection remained significantly lower (p < 0.05) in HF cows of treatment groups. However, the fertility of TEs was neither affected significantly (p > 0.05) by the superovulation protocol used nor by breed differences among donor cows. In conclusion, using CIDR-GnRH or CIDR-EB along with conventional superovulation protocol may enhance the efficiency of MOET programs in cattle. Furthermore, XB donor cows demonstrated a better performance than HF donor cows under subtropical conditions.

Ovarian superstimulation with FSH in Wagyu breed bovine donors impacts folicular dynamics but does not improve the amount of embryos

The increase of the world population generates the need to raise the production of food of vegetal and animal origin. In Brazil, livestock farming has evolved, demonstrating the important role of the country in the production of food. In this way, researchers of this productive chain have been looking for technologies related to production and reproduction, above all, to the use of reproduction biotechnologies, seeking to increase the production of different bovine breeds. Thus, the objective of this research was to investigate the efficacy of ovarian FSH super stimulation in bovine Wagyu oocyte donor females on follicular dynamics and in vitro embryo production. Twelve Wagyu animals, aged 12 to 24 months, randomly distributed in a crossover design were used in two groups: Group 1= animals not stimulated with FSH and Group 2= animals stimulated with FSH. The follicular, oocyte and embryonic variables were evaluated. It was observed that ovarian overstimulation in Wagyu oocyte donor cows with FSH improved the mean and large follicles but reduced the rate of oocyte recovery and, despite the best percentage of viable oocytes, there was no improvement in the amount of embryos produced in vitro.

Physiological Parameters to Identify Suitable Blood Donor Cows for Preparation of Platelet Rich Plasma

Platelet rich plasma (PRP) has been shown to be beneficial in the treatment of bovine mastitis, with an action comparable to that of antibiotics. Autologous treatment is feasible in experimental conditions but is difficult to apply in field conditions, particularly in acute mastitis. The ideal scenario would be to have heterologous PRP stored on every farm so that it is readily available when needed. In this paper, we analysed data collected during bovine mastitis treatment with heterologous PRP produced by casual donor cows on several farms. We tried to identify parameters which might be useful to identify the most suitable cows to be used as blood donors, to obtain the highest yield of PRP. Variables considered for each animal were the age, the parity, the date of the last parturition, the season of blood collection, the site of blood collection (jugular or mammary vein) and the reproductive status e.g., pregnant or not pregnant. There were statistically significant differences for all the variables considered from the 135 blood cows, except for the blood collection season. The highest yield of PRP was associated with nonpregnancy blood collection within three months of parturition, parity 3 or 4, and blood collection from the mammary vein.

BIOLOGICALLY ACTIVE PREPARATIONS USE IN THE SCHEME FOR STIMULATING PMSG «FOLLIGON» SUPEROVULATION IN DONOR COWS

The purpose of scientific research was to study the effect of the developed biologically active drug «Stimulin» on the level of superovulatory treatment with PMSG «Folligon» and the quantitative yield of the number of transferable embryos. The experiments were conducted on donor cows of the Ukrainian Black-and-White breed. Experimental donor cows were intramuscular administration injected for 8, 9, 10, 11 days of the oestrous cycle by drug «Stimulin» in a single dose 20 ml. Control animals in these days were similarly administered 20 ml of physiological saline solution. It was established that, when gonadotropin PMSG “Folligon” of a polyovulatory reaction in donor cows, receiving an intramuscular administration of the drug "Stimulin" dose of 20 ml from day 8 to 11 after estrus , promotes a positive trend until its level and the number of embryos suitable for transplantation increase (by 28,5 %), and also reduces neovulated follicles by 27,5%. With induction of gonadotrophin PMSG“Folligon” by superovulation in donor cows, receiving an intramuscular administration from day 8 to day 11 after estrus of the drug "Stimulin", contributes to the tendency to increase the level of superovulation and the number of transferable embryos and reduces the number of non-ovulated follicles.