​Effect of Season on Quantity and Competence of Oocytes Recovered Transvaginally from Holstein Cows for In vitro Fertilization

Background: Season of the year can affect the reproductive behavior in Holstein cows, altering the competition of the oocytes, reflecting a reduced production of embryos. The objective of this study was to evaluate the average of total oocytes, competition of oocytes and embryos in the in vitro production process at different season of the year in Holstein cows. Methods: During the four seasons of the year, was performed on each of the oocyte donor cows (winter, n = 957; spring, n = 1571; summer, n = 1776; autumn, n = 1128), by in vivo transvaginal follicular aspiration technique after the collection were subjected to the embryos production in vitro. Result: The highest number of total embryos were produced in winter and autumn, compared to spring and summer (3.76±0.16 and 3.54±0.18 vs. 2.73±0.11 and 2.45±0.10; respectively, P less than 0.05). During winter, a higher percentage of oocyte competition was observed, followed by autumn and spring and less competition shown in summer (26.03±0.39, 19.08±0.29, respectively, P less than 0.05). The quantity and competence of the oocytes collected and in vitro embryo production were drastically reduced during the hottest months of the year in this area of intense heat.

Asociación entre calidad de ovocitos recuperados y condición corporal en vacas criollas

Criollo high Andean cattle are of family economic importance for rural dwellers and contribute to food security. The objective of this study was to evaluate the quantity, quality, and nuclear status of ovarian oocytes according to the body condition of Criollo cows. Groups of cows were formed according to the body condition (1=emaciated, 5=obese): low (≤1.5), moderate (2 to 2.5) and high (≥3). Ovaries (n=212) were obtained from Criollo cows from the slaughterhouse. Oocytes were recovered by follicular aspiration classifying them into categories A, B, and C, placing a portion with ethanol-acetic acid (3:1) for 24 h, then stained with 2% Lacmoid. The nuclear status of the oocyte was evaluated, categorized as intact germinal vesicle (GV) and broken germinal vesicle (GVBD). The number of oocytes recovered per ovary among low, moderate, and high body condition cows were similar (p>0.05). There was an association between body condition and oocyte quality of category A (rs=0.49; P=0.001), B (rs=0.16; P=0.018); C (rs=-0.16; P=0.016). Higher percentages (p≤0.05) of oocytes of categories A and B were obtained in those of moderate and high body condition, as category C in low body condition. Body condition did not influence (p>0.05) oocyte nuclear status. No association was found (p=0.073) between body condition and oocytes with intact GV, nor with GVBD (p=0.737). It is concluded that body condition is associated with the quality of oocytes of category A, B and C, but not with the nuclear status of GV and GVBD in Criollo cows from the slaughterhouse.

In Vitro Production of Embryos from Prepubertal Holstein Cattle and Mediterranean Water Buffalo: Problems, Progress and Potential

Laparoscopic ovum pick-up (LOPU) coupled with in vitro embryo production (IVEP) in prepubertal cattle and buffalo accelerates genetic gain. This article reviews LOPU-IVEP technology in prepubertal Holstein Cattle and Mediterranean Water Buffalo. The recent expansion of genomic-assisted selection has renewed interest and demand for prepubertal LOPU-IVEP schemes; however, low blastocyst development rates has constrained its widespread implementation. Here, we present an overview of the current state of the technology, limitations that persist and suggest possible solutions to improve its efficiency, with a focus on gonadotropin stimulations strategies to prime oocytes prior to follicular aspiration, and IVEP procedures promoting growth factor metabolism and limiting oxidative and endoplasmic reticulum stress.

Collection and in vitro maturation of Mazama gouazoubira (brown brocket deer) oocytes obtained after ovarian stimulation

Summary In vitro production of embryos has gained prominence as a tool for use in wildlife conservation programmes in situ and ex situ. However, the development of this technique depends on steps that include ovarian stimulation, collection and oocyte maturation. The purpose of this study was to assess the feasibility of an ovarian stimulation protocol for follicular aspiration, the efficiency of videolaparoscopy for follicular aspiration and test a medium for in vitro oocyte maturation for the species Mazama gouazoubira. Five females were submitted to repeated ovarian stimulation (hormone protocol using controlled internal drug release), and estradiol benzoate on D0 and eight injections of follicle-stimulating hormone, once every 12 h, from D4 onwards at 30-day intervals. Fourteen surgical procedures were performed in superstimulated females, resulting in the collection of 94 oocytes and an average of 17.1 ± 9.1 follicles observed, 13.5 ± 6.6 follicles aspirated and 7.2 ± 3.7 oocytes collected per surgery. After collection, the oocytes were submitted to in vitro maturation for 24 h and stained with Hoechst 33342 dye to evaluate their nuclear status; 64.5% of the oocytes reached MII and 16.1% were spontaneously activated by parthenogenesis. The nuclear status of oocytes that did not undergo in vitro maturation was evaluated; 80.9% were found to be immature.

123 Efficiency of embryo production using ovum pickup oocytes recovered from stimulated and nonstimulated cows

Recovery of oocytes from live animals through ultrasound-guided transvaginal follicular aspiration (ovum pickup, OPU) is an essential element of modern livestock development. To increase the number of OPU oocytes retrieved, hormonal stimulation is broadly applied; however, the results are ambiguous. The aim of the present study was to compare recovery rate, quality of oocytes, and efficiency of invitro production of cattle embryos using OPU oocytes derived from hormonal treated and untreated cows. The study was performed in Simmental heifers at the age of 17 to 23 months. The heifers in the first group (n=7) were previously synchronized using prostaglandin F2α (Estrumate, MSD Animal Health) and gonadotrophin-releasing hormone (GnRH; Receptal, MSD Animal Health) and then underwent superstimulation with Stimofol (Reprobiol SPRL, Belgium) in a total amount of 0.25mg of FSH and 0.05mg of LH in a treatment of 6 injections on 3 consecutive days starting on Day −4 before OPU. The heifers of the second group (n=2) underwent OPU once a week for 5 weeks (10 OPU sessions). Groups of recovered cumulus–oocyte complexes (COCs) were matured in TCM-199 containing 10% fetal calf serum, 1mM Na-pyruvate, 50μg mL−1 gentamycin, 10μg mL−1 FSH, and 10μg mL−1 LH for 24h, fertilized in Fert-TALP (Tyrode’s-albumin-lactate-pyruvate) for 18 to 20h, and cultured in CR1aa medium for 7 days. All steps of IVP were performed at 38.5°C with 5% CO2 and 90% humidity. The cleavage and blastocysts rates were assessed on Day 2 and 7, respectively. In addition, Day-7 blastocysts were fixed, and the total cell number was determined using 4′,6-diamidino-2-phenylindole (DAPI) staining. The data were analysed by ANOVA using SigmaStat software package. As expected, the number of aspirated follicles per session in the first group of cows was higher than that in the second group: 14.6±1.2 vs. 6.8±0.5 (P<0.05). In total, 54 and 42 COCs were collected, which corresponds to recovery rates of 54.5±7.7 and 61.3±4.9% for stimulated and nonstimulated cows, respectively. After the quality evaluation, 36 COCs of SS heifers (67.2±1.9%) and 31 COCs of non-SS heifers (75.3±5.0%) were selected for IVP. We did not observe a significant difference in cleavage rate between two groups: 77.8% in the SS group vs. 71.0% in the non-SS group, whereas the rate of blastocyst production was higher (P<0.05) for SS heifers (25.0%) compared with non-SS animals (16.1%). The total number of cells in Day 7 blastocysts was significantly (P<0.01) higher in the first group (89.8±1.3) compared with the second group (70.6±3.2). Thus, superstimulation of heifers before OPU increases the efficiency of oocyte retrieval by ultrasound-guided transvaginal follicular aspiration as well as the developmental competence of OPU-derived bovine oocytes invitro. These studies were performed under financial support of the Russian Science Foundation (project No. 19-16-00115) and the Ministry of Science and Higher Education of Russia.

11 Effect of breed type on production of bovine embryos: Experience in Paraguay

One of the parameters that has the most influence on invitro embryo production programs is the number of oocytes obtained in each follicular aspiration session (ovum pickup; OPU). A significant impact of the breed of the donor on this parameter has been reported. The objective of this work was to compare the parameters of an invitro embryo production program of cows of different breeds in Paraguay. A total of 4811 OPU sessions were performed between 2016 and 2019 in cows classified according to breed and type. Beef-type breeds: Nelore (Bos indicus): n=1569; Aberdeen Angus (Bos taurus): n=1276; and Brangus (Cross): n=182 OPU, and Gyr (Bos indicus): n=1086; dairy breeds: Holstein (Bos taurus): n=401, and Girolando (Cross): n=297. The oocytes were aspirated and transported to the same laboratory located in Asunción, Paraguay. The invitro embryo production protocol was similar in all types. Bull semen tested for IVF was used. The number of oocytes, percentage viability, and percentage embryo production were evaluated. Additionally, the mean and standard deviation of the variables were calculated to show the efficiency of the processes, according to the production aptitude of the donors (meat or milk) and the breed type (Bos indicus, Bos taurus, or crosses). Continuous data were analysed with ANOVA and Tukey’s post hoc comparisons. Categorical data were analysed using Chi-squared at a significance level of 0.05. In this work, the production aptitude had no significant effect over the studied variables. However, some significant differences were found considering the breed type of the oocyte donor cow, as shown in Table 1. When crosses with the pure breeds are compared with their crosses, we found that Brangus produced more pregnancies and a greater number of oocytes/OPU compared with Angus (P<0.05). When the same comparison was made between Girolando and Holstein, no significant differences were found. The results show that Bos indicus breeds and their crosses have a greater capacity to produce more oocytes and embryos and that Bos indicus and crossbreeds were more efficient in producing pregnancies (2-fold) per OPU session than Bos taurus breeds. Additionally, crossing with meat indicus breeds generates better quality embryos and increases efficiency in the system of embryo production. Table 1. Comparison of embryo production parameters Item Breed type Bos indicus Cross Bos taurus Oocytes/OPU (mean±SD) 29.8±2.7a 26.3±7.8a 14.6±1.4b Viable oocytes, % 64.7±3a 64.6±3.4a 60.1±3.1a Embryo produced/OPU, % 35.2±4.7a 32.8±9.4a 26.2±4a Embryo/OPU 6.9±1.7a 5.4±1.7a 2.3±0.4b Pregnancy rate, % 29.6±5.5a 33.1±6.3a 25.3±5a Pregnancies/OPU 2.1±0.9a 1.9±1a 0.6±0.2b a,bValues within a row different letters differ (P<0.05).

144 Proof of concept for bilateral twin pregnancies in Trio allele carriers

Bovine twin birth is associated with detriments including increased embryo/fetal losses, malpresentation and dystocia. Incidence of these is lessened in bilateral versus unilateral twin pregnancy. This study was undertaken to assess use of follicular ablation by aspiration to create bilateral twin pregnancies in females with genetic potential for ~3.5 ovulations per cycle (Trio allele carriers). In Experiment 1, carriers (n = 30) and non-carriers (n = 10) were synchronized for ovulation and timed artificial insemination (TAI). Follicles (> 5 mm) in excess of one per ovary were aspirated ~16h preceding TAI. Follicle count for females with follicles on only one ovary was reduced to two. Blood was sampled 2 weeks post-TAI to assess progesterone (P4) concentrations; fetal count was determined by ultrasound 6 weeks post-TAI. P4 concentration post-TAI was significantly (P < 0.001) associated with both genotype and subsequent pregnancy status (pregnant non-carriers, 7.06 ± 0.68 ng/mL; pregnant carriers, 5.54 ± 0.55 ng/mL; non-pregnant non-carriers, 5.22 ± 1.05 ng/mL; non-pregnant carriers, 3.13 ± 0.42 ng/mL). Experiment 2 was undertaken to offset negative effects of follicular aspiration on subsequent P4 concentration observed in Experiment 1. Carriers (n = 38) and non-carriers (n = 32) were submitted to fixed-TAI and follicle ablation as described. Additionally, accessory corpora lutea were induced by administration of hCG (carriers) at d6 post-TAI. Consequently, P4 concentration post-TAI was significantly (P < 0.05) associated with subsequent pregnancy status (pregnant, 8.48 ± 0.61 ng/mL; non-pregnant, 6.70 ± 0.63 ng/mL) but not with genotype (carrier, 8.01 ± 0.59 ng/mL; non-carrier, 7.17 ± 0.64 ng/mL). Fetus number was greater in carriers (Exp1, 1.64 ± 0.81; Exp2, 1.45 ± 0.09) versus non-carriers (1.00 ± 0.00, both Experiments). Singles, twins, and triplets occurred in carriers in Experiment 1, whereas multiples in Experiment 2 were limited to twins. Genotype effects on pregnancy rate were not significant (P > 0.10) in either experiment. Results suggest follicular ablation to create bilateral twin pregnancies in Trio carriers is feasible but requires induction of accessory corpora lutea to offset the negative effects of follicular aspiration on subsequent P4 concentration.

Transmyometrial oocyte recruitment: a safe surgical procedure alternative to the transvaginal retrieval when ovaries are inaccessible

Background: This study aims to investigate the intra-cytoplasmic semen injection (ICSI) outcomes and the potential complications of transmyometrial oocyte retrieval performed in patients with one or both inaccessible ovaries.Methods: From January 2004 to December 2018 were evaluated 11,273 oocytes retrievals, of which 594 not conventional procedures for inaccessible ovaries through transvaginal retrieval. Despite alternative approaches tested by clinicians during the oocyte pick up (OPU), in 113 of these patients (Group A) were indispensable the needle’s passage through the myometrium, compared to as many controls (Group B) from the 481 remaining women without this necessity. The two resulting groups were matched for their reproductive outcomes and the relative developed complications.Results: No statistically significant differences were found between the two Groups in terms of number of eggs/embryos obtained, ongoing pregnancy rate and spontaneous miscarriage. A complete absence of complications as peritoneal and endometrial bleeding after 4 and 24 hours from the transmyometrial access was reported for the whole cohort.Conclusions: This study shows that the transvaginal transmyometrial oocyte retrieval does not affect the OPU outcome even if the passage of the needle occurs through the endometrium, not inducing significant complications. Thus, here we support both efficacy and safety of transmyometrial follicular aspiration as a valid surgical approach in conditions of inaccessible ovaries.

Practical application of an impractical bovine genotype: creating bilateral twin pregnancies in Trio allele carriers

Bovine twin birth is associated with detriments, including increased embryo/fetal losses, malpresentation, and dystocia. Incidence of these is lessened in bilateral compared with unilateral twin pregnancy. This study was undertaken to assess the use of follicular ablation by aspiration to create bilateral twin pregnancies in females with genetic potential for ~3.5 ovulations per cycle (Trio allele carriers). In experiment 1, carriers (n = 30) and noncarriers (n = 10) were synchronized for ovulation and timed artificial insemination (TAI). Follicles (>5 mm) in excess of one per ovary were aspirated ~16 h preceding TAI. Follicle count for females with follicles on only one ovary was reduced to two. Blood was sampled 2 wk post-TAI to assess progesterone (P4) concentrations; embryo count was determined by ultrasound 6 wk post-TAI. Circulating P4 concentration post-TAI was significantly (P < 0.001) associated with both genotype and subsequent pregnancy status (pregnant noncarriers: 7.06 ± 0.68 ng/mL; pregnant carriers: 5.54 ± 0.55 ng/mL; nonpregnant noncarriers: 5.22 ± 1.05 ng/mL; nonpregnant carriers: 3.13 ± 0.42 ng/mL). Experiment 2 was undertaken to offset the negative effects of follicular aspiration on subsequent P4 concentration observed in experiment 1. Carriers (n = 38) and noncarriers (n = 32) were submitted to TAI and follicle ablation as described for experiment 1. Additionally, accessory corpora lutea (CL) were induced in carriers by the administration of human chorionic gonadotropin (carriers) at day 6 post-TAI. Consequently, P4 concentration post-TAI was significantly (P < 0.05) associated with subsequent pregnancy status (pregnant: 8.48 ± 0.61 ng/mL; nonpregnant: 6.70 ± 0.63 ng/mL) but not with genotype (carrier: 8.01 ± 0.59 ng/mL; noncarrier: 7.17 ± 0.64 ng/mL). Embryo number was greater in carriers (exp. 1: 1.64 ± 0.81; exp 2: 1.45 ± 0.09) vs. noncarriers (1.00 ± 0.00, both experiments). Single, twin, and triplet pregnancies occurred in carriers in experiment 1, whereas multiples in experiment 2 were limited to twin pregnancies. Genotype effects on pregnancy rate were not significant (P > 0.10) in either experiment. Results suggest that follicular ablation to create bilateral twin pregnancies in Trio carriers is feasible but requires the induction of accessory CL to offset the negative effects of follicular aspiration on subsequent P4 concentration and associated fertility outcomes.