Ultrasound-guided Ovum Pick-up Research Articles

Effect of Hormones, Season and Lactation on Follicular Size, Oocyte Competency and Embryo Production in Sahiwal Cattle

Background: Transvaginal ultrasound-guided ovum pick-up (OPU) and in vitro embryo production (IVEP) techniques are widely used for enhancing reproductive efficiency. The number and quality of embryos have been reported to be influenced by various factors like source of hormones, season, lactation in cattle. Methods: Twenty-one multiparous, normal cyclic Sahiwal cows (aged: 6-8 years; lactating: n-11 and non-lactating: n-10) were selected randomly and the experiment was conducted during hot (April to September) and cold (October to March) seasons. All the donor cows were administered 10 µg GnRH at random stage of oestrous cycle followed by FSH (Folltropin-V™ or Stimufol) at the rate of 100, 60 and 40 mg after 48, 60 and 72 h of GnRH administration. After 24 hours of coasting period, follicular aspiration was performed by stabilizing the targeted follicles with the help of ultrasonography. The procedures of COCs grading, in vitro maturation, fertilization, followed by culture were performed as per the standard protocols. Results: The hormonal treatment was effective in improving the oocytes’ developmental competency and in vitro embryo production; with no statistical differences between the two sources. The follicle size distribution revealed a lower (P less than 0.05) medium-sized and small-sized follicular proportion during the hot season with a higher average count of dominant (large-sized) follicles. While, good quality (A+B+C) oocytes, matured and fertilized oocytes were significantly higher (P less than 0.05) in non-lactating donors, especially among cool-season collections.

Successful ultrasound-guided ovum pick-up (OPU) and subsequent in vitro embryo production in a domestic cat

Ovum Pick Up (OPU) is a minimally invasive technique widely used in cattle and mares for oocyte retrieval, involving ultrasound-guided puncture of ovarian follicles. It has been demonstrated that this technique is safe for its repeated use in the same female without affecting her reproductive health, allowing for the retrieval of oocytes in individuals regardless of their reproductive status. The oocytes obtained through OPU can subsequently be used for in vitro embryo production (IVP) using assisted reproductive techniques (ARTs) or be cryopreserved in biobanks for their future use. Traditionally, the minimally invasive technique of choice performed in vivo in domestic and wild felines was LOPU (laparoscopic-guided ovum pick up). The present study was designed to explore if ultrasound-guided OPU in the domestic cat is safe and effective. In an initial series of ex vivo experiments (n = 92 ovaries, n = 434 oocytes), the effect of different aspiration pressures for oocyte collection was explored. These experiments identified 43 mmHg as the optimal aspiration pressure, resulting in the highest recovery rate and a favorable maturation and blastocyst rate. Subsequently, 16 grade I and II oocytes were retrieved by OPU and 101 oocytes were retrieved following ovariectomy and slicing. Sixteen oocytes obtained with each technique were subjected to in vitro maturation (IVM) and in vitro fertilization (IVF). A total of 14 presumptive zygotes were selected for in vitro culture (IVC) from each group (OPU and slicing), obtaining a cleavage rate of 57.1 % and 64.2 %, a morula rate of 28.5 % in both groups, and a blastocyst rate of 7.14 % and 14.2 % respectively. The hormonal stimulation protocol was well-tolerated, with no adverse effects observed. Moreover, no complications arose during the ovariectomy performed post-OPU. The use of this technique in domestic cats represents a significant step forward in terms of safety, replicability, and invasiveness, serving as a valuable model for its application in wild felids species. Additional research involving a greater number of animals is required to validate these encouraging findings.

Ovum Pick Up and in vitro Embryo Production using Sex Sorted Semen in Cows

Background: Ovum pick-up, a technique for extracting immature oocytes from antral follicles, with in vitro embryo production, is a way to produce large number of embryos from live donors. Methods: By transvaginal ultrasound-guided ovum pick-up, oocytes were collected from large (9-19 mm), medium (4-8 mm) and small (2-3 mm) ovarian follicles of donor cows (n=8). The recovered oocytes were classified as A, B, C, D and E grades. Oocytes were subjected toin vitro maturation, fertilization (with sex sorted semen) and culture. Cleavage and blastocyst rates were recorded on day 4 and day 7 of culture respectively. Result: Total of 278 follicles were aspirated. The follicles aspirated per cow per OPU session was 12.67±1.86. The oocyte recovery rate was 45.31±7.35. Mean percentage of grade A, B, C, D and E oocytes was 15.69±3.91, 24.97±3.02, 37.53±3.45, 19.44±3.76 and 2.38±2.38 respectively. In vitro maturation rate (IVM%) was 91.71±2.38% and fertilization rate was 90.18±1.52%. Cleavage and blastocyst rates observed was 55.1±5.90 and 24.81±2.95% respectively. The mean percentages of different grades of embryos observed was, code 1: 21.13±2.78, code 2: 14.41±3.25, code 3: 19.56±4.11 and code 4: 44.9±5.90%. Transvaginal ovum pick up can be performed repeatedly without hormonal stimulation. Sex sorted semen can be used for in vitro production of female embryos.

Experiences with transvaginal Ovum Pick-Up (OPU) in sows

Transvaginal ultrasound-guided Ovum Pick-Up (OPU) is an established technique in other species. Due to several challenges, there are few publications addressing the procedure in sows. An efficient OPU technique may allow for the collection of numerous oocytes from valuable sows for porcine in vitro embryo production, gene editing and cloning programmes, or cryopreservation. We aimed to improve transvaginal OPU and equipment for this technique in sows. In experiment 1, we conducted 13 OPU sessions on three Landrace x Large White hybrid sows under general anaesthesia, while the second experiment explored OPU in non-sedated animals (N = 6) physically restrained in a commercial claw trimming chute. The experiments resulted in 6.6 ± 5.6 (mean ± SD) and 7.7 ± 8.9 recovered cumulus-oocyte complexes per session, respectively. Post-mortem examination of the pelvic and abdominal cavities of the three sows subjected to repeated OPU sessions did not reveal major acute or chronic pathological lesions. The only sow which was inseminated after the experiment delivered 16 liveborn piglets at term. Salivary cortisol levels increased during the procedure in non-sedated and physically restrained sows but returned to baseline 1 h later (n = 5), indicating a short-term stress response. The described OPU technique and equipment have the potential to retrieve considerable numbers of oocytes by repeated procedures on valuable mature sows. Follow-up studies are needed to optimize the efficiency of the aspiration of high-quality oocytes and to describe the developmental competence of these OPU-derived oocytes. It is also essential to further investigate sow welfare during and after the procedure before recommending porcine transvaginal OPU as a sustainable and welfare-friendly procedure.

Proteome of follicular fluid from Murrah buffaloes (Bubalus bubalis): Normal cycling versus repeat-breeder females

Clinical evaluations have shown that repeat breeding is a major cause of infertility in buffaloes. The follicular fluid (FF) composition reflects directly metabolic status and fertility in females. Given this scenario, this study aimed to perform quantitative proteomic analysis of the FF collected from normal cycling (NC) and compared to repeat-breeder females. According to farm records, buffaloes were divided into two groups: normal cycling (NC, n = 7) and repeat-breeder (RB, n = 8) females. After estrus synchronization and using ultrasound-guided ovum pick-up, FF was aspirated from large follicles (˃ 8 mm). Posteriorly, proteins were identified by the shotgun method. A total of 119 proteins were identified and, among theses, three were uncharacterized and a protein (LOC123334375) was identified only in the NC group. The protein HP-25 homolog 2 was expressed only in RB females. The LFQ (label-free quantitation)-intensity of the proteins afamin (AFM), transthyretin (TTR), clotting factor IX (F9) and Xaa-Pro dipeptidase (PEPD) was significantly (P < 0.05) higher in RB than in NC females. In conclusion, the use of quantitative proteomics proved to be an important tool for the study of RB in buffaloes. The identification of HP-25 homolog 2 protein only in the RB females suggests that it can be used as a biomarker for this reproductive disorder. Additionally, an uncharacterized (LOC123334375) protein was identified only in normal cycling females and compensatory oxidative stress proteins were more expressed in repeat-breeders.

Integration analysis of metabolome and transcriptome reveals the effect of arachidonic acid on hemorrhagic anovulatory follicles in jennies

Compared with other livestock, fertility is low in jennies. Although the majority of jennies shows normal ovulation throughout the year, some jennies display hemorrhagic anovulatory follicles (HAF) in the middle of the winter or summer. Based on our previous results (https://doi.org/10.1111/rda.13883), this research was carried out in from November to January of the next year. In experiment 1, a total of 24 Yangyuan jennies (4∼8 years old) were included, with half of them showing normal preovulatory follicles (control group, Con-G) while the other half had HAF ad diagnosed by transrectal ultrasound (HAF-G). The content of the preovulatory follicle or HAF was collected from each jenny by ultrasound-guided ovum pick-up. The follicular fluid (FF) and granulosa cells (GCs) were used for omics analysis. The results of metabolomics analysis showed that the level of arachidonic acid (AA) was significantly increased in HAF-G (P < 0.05). According to transcriptomics results, the mRNA levels of arachidonate 5-lipoxygenase gene (ALOX5) was significantly increased (P < 0.05) while the level of cytochrome P450 family 2 subfamily J member 2 gene (CYP2J2) was significantly decreased in HAF-G (P < 0.05). However, there was no significant difference between the two groups in the expressionsof prostaglandin-endoperoxide synthase 1 gene (COX1) and COX2 (P > 0.05). Meanwhile, the expressions of FSHR, LHCGR and CYP19A1 were significantly decreased in the HAF-G compared with the Con-G (P < 0.05). In order to pursue the molecular mechanism of AA regulating the functions of granulosa cell, the experiment two was carried out. The mice GCs from antral follicles were cultured in media supplemented with 0, 50 μM or 100 μM AA respectively, and both of cell viability and some genes’ expressions were assayed. The results demonstrate that the survival of GCs and mRNA expressions of FSHR and LHCGR were higher in 50 μM AA group than the control group (P < 0.05). However, when the AA concentration was increased to 100 μM in the medium, the survival rates and expression of FSHR and LHCGR were decreased in comparison to the control group (P < 0.05). Moreover, 100 μM AA increased the expression of ALOX5 and decreased the expression of CYP2J2 compared with 50 μM AA, which was similar to GCs from HAF of jennies. In conclusion, the occurrence of HAF in jennies may be related to the accumulation of AA in follicular fluid of the preovulatory follicles which caused abnormal expression of FSHR, LHCGR and ALOX5 on GCs leading to anovulation and HAF.

Experimentally Induced Endometritis Impairs the Developmental Capacity of Bovine Oocytes†.

Uterine infection is associated with infertility in women and dairy cows, even after the resolution of infection. However, the mechanisms causing this persistent infertility are unclear. Here, we hypothesized that induced endometritis in non-lactating dairy cows would reduce the developmental competence of oocytes. Non-lactating Holstein cows received an intrauterine infusion of endometrial pathogenic bacteria (Escherichia coli and Trueperella pyogenes; n=12) or vehicle control (n=11) on day 2 of the estrous cycle. Bacterial infusion increased expression of endometrial inflammatory mediators, and a mucopurulent discharge in the vagina confirmed the establishment of endometritis. Oocytes were collected by transvaginal ultrasound-guided ovum pickup on days 2, 24, 45, and 66 following infusion and subjected to in vitro fertilization and embryo culture. Bacterial infusion resulted in fewer cleaved oocytes developing to morulae compared to vehicle-infused controls (30.7 versus 45.0%), with the greatest effect observed in oocytes collected on day 24. Development to morula was inversely correlated with endometrial expression of IL6 on day 6. The expression of genes associated with embryo quality did not differ significantly between morulae from bacteria-infused and control cows. Artificial insemination 130days after intrauterine infusion resulted in normal, filamentous embryos that produced interferon tau 16days after conception in both infusion groups. This model of experimentally induced uterine infection successfully resulted in endometritis and a reduction in the proportion of oocytes that developed to morulae following in vitro fertilization. In conclusion, endometritis reduced the capacity of oocytes to develop to morulae.

13 Invitro and invivo embryo production during foal heat in a mare: A case report

The objective of this case report is to describe invitro and invivo embryo production during foal heat in a mare. A 16-year-old American Quarter Horse mare was presented for breeding and nonsurgical embryo recovery days post-foaling. Upon transrectal ultrasonographic examination, the donor mare was determined to be in oestrous (foal heat) with presence of moderate uterine oedema, a relaxed cervix, absence of uterine fluid, a 35-mm dominant follicle, and multiple (n=7) subordinate follicles (10-20mm). To increase embryo production, oocytes were retrieved by ultrasound-guided ovum pickup of all subordinate follicles. Upon collection, oocytes (n=4) were placed in embryo holding medium for 18h at room temperature (22°C) and then matured in equine maturation medium (EqMat, Campos-Chillon et al. 2019 J. Equine Vet. Sci. 73, 51-55) for 30h at 38.5°C in 6% CO2, 5% O2, and 89% N2. Matured oocytes (n=3), as assessed by extrusion of the first polar body, underwent intracytoplasmic sperm injection with frozen-thawed semen and a Piezo-driven injection system. Injected oocytes were cultured in SCF1 (Owen et al. 2017 Reprod. Fertil. Dev. 29, 129-130) for 5 days and Dulbecco's modified Eagle medium/F12 plus 5% FBS for 2 days at 38.5°C in 6% CO2, 5% O2, and 89% N2. On Day 10 post-foaling, the dominant follicle was 45mm and the donor mare was inseminated with fresh semen (500×106 progressively motile spermatozoa) and ovulation was induced with 1.8mg of deslorelin IM (SucroMate, Thorn BioScience LLC). The donor mare ovulated within normal limits by 48h post-induction of ovulation (12 days post-foaling) and embryo recovery was attempted 8 days post-ovulation (20 days post-foaling). The donor mare was then administered 250μg of Cloprostenol IM (Estrumate, Merck Animal Health) to induce oestrus and was inseminated again with cooled semen (1×109 progressively motile spermatozoa) for two consecutive cycles. Maturation, cleavage, and blastocyst rates were 57% (4/7), 67% (2/3), and 33% (1/3), respectively. The nonsurgical embryo recovery resulted in one expanded blastocyst grade 1. Both invitro- and invivo-produced blastocysts were transferred into synchronized recipients 5 days after oestrus on Day 16 and 20 post-foaling respectively. The donor mare got pregnant after the second cycle post-embryo recovery attempt. Pregnancy status of recipients and donor mare were evaluated at 14, 21, 30, and 50 days after embryo transfers or insemination with normal development for the three pregnancies. In conclusion, to our knowledge, this is the first case report describing invitro and invivo embryo production performed during foal heat increasing number of pregnancies in a post-foaling mare in a single cycle.

Production of piglets from in vitro-produced blastocysts by ultrasound-guided ovum pick-up from live donors

The objective of this research was to develop a system for piglet production by transvaginal ultrasound-guided ovum pick-up (OPU), in vitro production (IVP) of embryos and embryo transfer. First, to establish a culture system for a small number of oocytes or embryos, we evaluated the effect of different incubation volumes and culture densities on fertilizing ability and developmental competence in vitro. Porcine oocytes derived from slaughterhouse ovaries were matured, fertilized and then cultured in vitro in groups as follows: 50 oocytes in 500 μL medium for IVM, 20 oocytes in 100 μL medium for IVF and 20 embryos in 40 μL medium for IVC (Group I); 20 in 100 μL for IVM, 20 in 100 μL for IVF and 20 in 40 μL for IVC (Group II); and 10 in 100 μL for IVM, 10 in 100 μL for IVF and 10 in 40 μL for IVC (Group III). Percentages of sperm penetration, cleavage and blastocyst formation did not differ among the groups. Second, to increase the collection efficiency of porcine oocytes by transvaginal ultrasound-guided OPU, the effects of aspiration pressure on follicular oocyte collection were assessed. Oocytes were aspirated from ovaries of live sows using 80 or 100 mmHg. The recovered oocytes were divided into four categories according to the surrounding cumulus cells and quality of oocytes. The number of oocytes recovered using 100 mmHg pressure was significantly higher than with 80 mmHg pressure. However, there were no significant differences in the population of oocytes grouped by the morphological criteria, number of blastocysts per session and the total cell number in blastocysts between the two vacuum pressures. Finally, 81 oocytes obtained by OPU from five donor sows were subjected to IVP and 47 transferable embryos (9.4 ± 4.0 [mean ± SD] morulae/blastocysts per session) were obtained at 5 days after IVF. When they were transferred into five recipient gilts (5–16 embryos per recipient), three of five recipients became pregnant and farrowed a total of 12 live piglets. The present results demonstrate that porcine blastocysts can be produced by OPU-IVP and develop to full term after embryo transfer.

Supplementation of cows with linseed: effects on antral follicle count, oocyte quality, and nutritional and plasma metabolite parameters

Linseed is distinguished by its composition of fatty acids, which are rich in unsaturated acids, and by the slow release of oil in the rumen, consequently providing a greater amount of lipids of interest for intestinal absorption. The aim of this study was to evaluate the effect of linseed supplementation on the antral follicle count (AFC) and oocyte quality of Girolando cows, as well as their digestive parameters and plasma metabolite. Twelve Girolando cows were used in the study and were randomly distributed in two experimental groups: control (CTL, n = 6) and linseed (LINS, n = 6). During the 126 days of supplementation, seven transvaginal ultrasound-guided ovum pick-up (OPU) sessions were performed at intervals of 21 days. AFC was performed in each OPU session. The cows fed with linseed showed no significant difference in the dry matter coefficient of digestibility (63.11 and 62.88), crude protein (62.7 and 55.26), neutral detergent fiber (NDF; 75.36 and 77.15), or acid detergent fiber (ADF; 72.45 and 74.77) for LINS and CTL, respectively. On the contrary, a higher level of ethereal extract was observed for LINS (69.31 vs. 40.7). There was no effect (p &gt; 0.05) of lipid supplementation on plasma metabolite between groups and OPU sessions. There was no difference (p &gt; 0.05) in the mean number of antral follicles (CTL: 31.14 ± 1.97; LINS: 25.52 ± 2.68), cumulus oocyte complexes recovered (CTL: 6.45 ± 1.66; LINS: 5.28 ± 1.18), or oocyte quality (CTL: 60.48% ± 8.46; LINS: 64.54% ± 7.77). The supplementation of 800g of linseed in the diet of Girolando cows did not alter the apparent digestibility of nutrients, AFC, or quality of oocytes obtained by OPU.

Effects of follicle-stimulating hormone followed by gonadotropin-releasing hormone on embryo production by ovum pick-up and in vitro fertilization in the river buffalo (Bubalus bubalis).

In this study, we examined the effects of superstimulation using follicle‐stimulating hormone (FSH) followed by gonadotropin‐releasing hormone (GnRH) on buffalo embryo production by ultrasound‐guided ovum pick‐up (OPU) and in vitro fertilization (IVF). Nine Murrah buffaloes were subjected to OPU‐IVF without superstimulation (control). The morphologies of the oocytes collected were evaluated, and oocytes were then submitted to in vitro maturation (IVM). Two days after OPU, same nine buffaloes were treated with twice‐daily injections of FSH for 3 days for superstimulation followed by a GnRH injection. Oocytes were collected by OPU 23–24 hr after the GnRH injection and submitted to IVM (the superstimulated group). The total number of follicles, number of follicles with a diameter > 8 mm, and number of oocytes surrounded by multi‐layered cumulus cells were higher in the superstimulated group than in the control group (p ≤ 0.05). After IVF, the percentages of cleavage and development to blastocysts were higher in the superstimulated group than in the control group (p < 0.05). In conclusion, superstimulation improved the quality of oocytes and the embryo productivity of OPU‐IVF in river buffaloes.

Plasma anti-Müllerian hormone as a predictive endocrine marker to select Bos taurus (Holstein) and Bos indicus (Nelore) calves for in vitro embryo production

This study evaluated the association between plasma anti-Müllerian hormone (AMH) concentrations and in vitro embryo production in Bos indicus (Nelore; experiment 1) and Bos taurus (Holstein; experiment 2) calves superstimulated or not with 140 mg of porcine follicle-stimulating hormone (pFSH; 4 decreasing doses twice daily). Oocytes were recovered from calves aged 2 to 4 mo after receiving gonadotropin stimulation (Nelore, n = 15; Holstein, n = 12) or not (Nelore, n = 15; Holstein, n = 12). Cycling heifers formed a positive control group (n = 15 for Nelore [aged 18–24 mo], n = 10 for Holstein [aged 14–16 mo]). All the calves underwent laparoscopic ovum pickup, and cycling heifers underwent a regular transvaginal ultrasound-guided ovum pickup for oocyte recovery. Immediately before oocyte retrieval, blood samples were taken for subsequent AMH determination (ng/mL). Regardless of the genetic group, calves that received pFSH (3.6 ± 1.1 in Nelore and 4.6 ± 1.2 in Holstein) or did not receive pFSH (3.2 ± 1.0 in Nelore and 2.5 ± 0.8 in Holstein) had greater plasma AMH concentrations (P = 0.01 in Nelore and P = 0.003 in Holstein) than cycling heifers (1.1 ± 0.2 in Nelore and 0.6 ± 0.07 in Holstein). AMH concentrations in calves with or without pFSH were similar in both genetic groups (3.6 ± 1.1 vs 3.2 ± 1.0 in Nelore; 4.6 ± 1.2 vs 2.5 ± 0.8 in Holstein). In calves, positive correlations were observed between plasma AMH concentrations and the numbers of follicles >2 mm (r = 0.86, P < 0.0001 in Nelore; r = 0.78, P < 0.0001 in Holstein), cumulus-oocyte complexes (COCs) retrieved (r = 0.91, P < 0.0001 in Nelore; r = 0.82, P < 0.0001 in Holstein), COCs cultured (r = 0.71, P < 0.0001 in Nelore; r = 0.79, P < 0.0001 in Holstein), and blastocysts produced (r = 0.62, P = 0.0003 in Nelore; r = 0.58, P = 0.009 in Holstein), and these results were independent of pFSH treatment. In conclusion, calves had greater plasma AMH concentrations than cycling heifers. In addition, treatment with pFSH did not influence AMH concentrations in calves, regardless of the genetic group. More importantly, plasma AMH concentrations were positively correlated with the antral follicle population and the number of COCs retrieved, COCs cultured, and blastocysts produced in B indicus and B taurus calves. Therefore, AMH is a promising tool for selecting oocyte donor calves to maximize results during in vitro embryo production.

Effects of a high-energy diet on oocyte quality and in vitro embryo production in Bos indicus and Bos taurus cows

The effects of different dietary energy levels [100 and 170% for maintenance (M) and high energy (1.7M), respectively] on metabolic, endocrine, and reproductive parameters were evaluated in nonlactating Bos indicus (Gir; n=14) and Bos taurus (Holstein; n=14) cows submitted to ultrasound-guided ovum pick-up followed by in vitro embryo production. The oocyte donor cows were housed in a tiestall system and fed twice daily (0800 and 1600 h). Twenty-one days before the beginning of the experiment, the animals were fed with a maintenance diet for adaptation followed by the experimental diets (M and 1.7M), and each cow underwent 9 ovum pick-up procedures 14 d apart. The recovered oocytes were cultured in vitro for 7 d. We measured glucose and insulin concentrations and performed glucose tolerance tests and the relative quantification of transcripts (PRDX1, HSP70.1, GLUT1, GLUT5, IGF1R, and IGF2R) from the oocytes recovered at the end of the experimental period. No interactions were observed between the effects of genetic groups and dietary energy level on the qualitative (viable oocytes, quality grade, and oocyte quality index) and quantitative (oocytes recovered) oocyte variables. There were no effects of dietary energy level on the qualitative and quantitative oocyte variables. However, Bos indicus cows had greater numbers of recovered structures, viable oocytes, and A and B oocyte grades as well as better oocyte quality index scores and lower DNA fragmentation rates compared with Bos taurus donors. In vitro embryo production (cleavage and blastocyst rates and number of embryos) was similar between diets, but the 1.7M diet reduced in vitro embryo production in Bos indicus cows after 60 d of treatment. Moreover, Bos indicus cows on the 1.7M diet showed lower transcript abundance for the HSP70.1, GLUT1, IGF1R, and IGF2R genes. All cows fed 1.7M diets had greater glucose and insulin concentrations and greater insulin resistance according to the glucose tolerance test. In conclusion, increasing dietary energy did not interfere with oocyte numbers and quality, but the 1.7M diet reduced in vitro embryo production in Bos indicus cows after 60 d of treatment. Finally, Bos indicus cows had greater oocyte quality, greater numbers of viable oocytes and greater in vitro embryo yield than Bos taurus.

257 IN VITRO YAK EMBRYO PRODUCTION THROUGH CONVENTIONAL AND OVUM PICKUP METHODS

Yak is one of the most important economically useful animals for highlanders. The decline in the yak population demands effective measures for conservation and multiplication of elite germplasm. In vitro production of embryos and their cryopreservation and transfer to suitable recipients for production of elite calves may contribute to fulfill the objectives. The work was conducted at the National Research Center on Yak over a period of 3 years. The ovaries of slaughtered animals were used for collecting oocytes through aspiration of follicles followed by slicing of ovaries in the conventional method. Trials were conducted using 7 cyclic parous yaks for ultrasound-guided ovum pickup (OPU) at Nyukmadung farm (2700 m above mean sea level). The technique followed was similar to that in buffaloes with slight modification. Categories of oocytes classified A (2–3 layers of cumulus) and B (at least one layer of cumulus) obtained through the processes were subjected to in vitro maturation using standardized maturation medium (TCM-199 + 10% follicular fluid + sodium pyruvate + l-glutamine + 10% heat inactivated oestrus cow serum + pFSH + 17β oestradiol). The frozen-thawed yak sperm were capacitated using the swing-up method before their incubation with matured oocytes using BO medium. Oocytes matured for 24 h were washed 5 to 6 times with BO medium and then co-incubated with in vitro capacitated spermatozoa (0.1 to 0.25 million) for fertilization (8–10 oocytes per group) in 100-µL droplets of BO medium under mineral oil in 35-mm Petri dishes and placed in a CO2 incubator (5% CO2, 90% RH) at 38.5°C for 16 to 18 h. The presumed zygotes were washed several times in mCR2aa (modified Charles Rosenkrans) washing medium and then cultured in culture medium for 7 days on original beds of granulosa cells. The rates of maturation and fertilization of oocytes collected by conventional and OPU technique were comparable (Table 1). This may be attributed to greater numbers of good quality oocytes recovered in the conventional method. Embryos developed up to the stage of compact morula and blastocysts (24.66% through conventional and 22.73% through OPU) were cryopreserved using the vitrification method for further study. Thirteen embryos were transferred non-surgically to one each of 13 yak recipients; 5 became pregnant and only 1 recipient transferred with a cryopreserved-thawed embryo, developed through OPU, delivered one male calf, leading to the first successful production of an IVF yak calf in the world. The present findings are suggestive of using the OPU technique for in vitro embryo production, though resulting in lower numbers of transferable embryos (Table 1), because availability of ovaries for conventional IVF is a major constraint in yak. Table 1.Comparative in vitro yak embryo production rate with recovery of oocytes by conventional or ovum pickup (OPU) method

Minimizing the Risk of Infection and Bleeding at Trans-Vaginal Ultrasound-Guided Ovum Pick-up: Results of a Prospective Web-Based World-Wide Survey.

The objective of this study was to identify clinical practices worldwide, which would help in recognizing women at risk of excessive bleeding or of developing pelvic infection following trans-vaginal ovum pick-up (TV-OPU), measures taken to minimize risks and their management. A prospective, web-based questionnaire with distinct questions related to the practice of TV-OPU. A total of 155 units from 55 countries performing 97,200 IVF cycles annually responded to this web-based survey. A majority (65%) responded that they would routinely carry out full blood count, while 35% performed coagulation profile. Less than a third agreed screening women for vaginal infections. About a third used both sterile water and antiseptic to minimize ascending infection, and 52% used antibiotics for prophylaxis. Doppler ultrasound was routinely used by 20% of clinicians. 73% of the clinicians preferred conservative management as their first line management for patients diagnosed with intra-abdominal bleeding. The study has identified a wide variation in the practices of minimizing infection and bleeding complications. The dearth of good quality evidence may be responsible for the lack of published guidelines, and therefore a lack of consensus on the optimum practice for minimizing the risk of infection and bleeding during TV-OPU.

Longitudinal Study of Reproductive Performance of Female Cattle Produced by Somatic Cell Nuclear Transfer

The objective of this study was to determine whether or not reproductive performance in cattle produced by somatic cell nuclear transfer (SCNT) is significantly different from that of their genetic donors. To address this question, we directed two longitudinal studies using different embryo production procedures: (1) superovulation followed by artificial insemination (AI) and embryo collection and (2) ultrasound-guided ovum pick-up followed by in vitro fertilization (OPU-IVF). Collectively, these two studies represent the largest data set available for any species on the reproductive performance of female clones and their genetic donors as measured by their embryo production outcomes in commercial embryo production program. The large-scale study described herein was conducted over a six-year period of time and provides a unique comparison of 96 clones to the 40 corresponding genetic donors. To our knowledge, this is the first longitudinal study on the reproductive performance of cattle clones using OPU-IVF. With nearly 2,000 reproductive procedures performed and more than 9,200 transferable embryos produced, our observations show that the reproductive performance of cattle produced by SCNT is not different compared to their genetic donors for the production of transferable embryos after either AI followed by embryo collection (P = 0.77) or OPU-IVF (P = 0.97). These data are in agreement with previous reports showing that the reproductive capabilities of cloned cattle are equal to that of conventionally produced cattle. In conclusion, results of this longitudinal study once again demonstrate that cloning technology, in combination with superovulation, AI and embryo collection or OPU-IVF, provides a valuable tool for faster dissemination of superior maternal genetics.

Acid–Base Parameters and Steroid Concentrations in Pre‐Ovulatory Follicles and Plasma of Lactating Dairy Cows with Spontaneous and Synchronized Oestrus or Follicular Cyst

The study aimed to compare the acid-base balance and steroid concentrations between follicular fluids (FF) of pre-ovulatory follicles derived from a spontaneous oestrus (SO), synchronized or induced oestrus (IO) and follicular cysts (CYS) and between FF and blood in dairy cows. Forty-two dairy cows were included in this study. The animals were allocated to three groups: SO (n=23); IO (n=11) using GnRH at day 0 and day 9 and PGF2 α at day 7; and animals with CYS (n=10). The follicular fluids (FF) were aspirated from the cyst/pre-ovulatory follicles (∅≥15mm) after SO and after second GnRH dose in IO by transvaginal ultrasound-guided ovum pick-up technique. Blood samples (BL) were collected in heparinized vacutainer tubes. The oxygen tension (pO2) in FF of IO was higher (p<0.05) than in SO and CYS groups. There were negative correlations (p<0.001, r=-0.89) between FF and blood pO2. The carbon dioxide tension (pCO2) and lactate level in FF of CYS group were higher (p<0.05) than in SO and IO groups. There were negative correlations (p<0.01, r=-0.73) between blood and FF pO2. Oestradiol-17β concentration in pre-ovulatory follicles and plasma of the SO group was higher (p<0.001) than in IO and CYS groups. Progesterone concentration in pre-ovulatory follicles and plasma of the SO and IO groups was lower (p<0.01) than in CYS group. Plasma androstenedione concentration in SO and IO groups was higher (p<0.05) than in CYS group. In conclusion, acid-base parameters, E2 and P4 levels in the follicular fluid of both IO and CYS groups were deviated greatly from the physiological level (disturbances of intrafollicular/intracystic environment), which may affect the quality of both the oocyte and the granulosa cells.

Single nucleotide polymorphisms in the bovine genome are associated with the number of oocytes collected during ovum pick up

The number of follicles recruited in each estrous cycle has gained practical importance in artificial reproductive technology, as it determines the oocyte yield from ultrasound-guided ovum pickup for in vitro embryo production. We aimed to identify single nucleotide polymorphisms (SNPs) in bovine genes related to reproductive physiology and evaluate the association between the candidate SNPs and the number of oocytes collected from ultrasound-guided ovum pickup. We sequenced genomic segments of GDF9, FGF8, FGF10 and BMPR2 and identified seventeen SNPs in the Bos taurus and Bos indicus breeds. Two SNPs cause amino acid changes in the proteins GDF9 and FGF8. Three SNPs in GDF9, FGF8 and BMPR2 were genotyped in 217 Nelore cows (B. indicus), while two previously identified mutations in LHCGR and mitochondrial DNA (mtDNA) were genotyped in the same group. The polymorphisms in GDF9, FGF8, BMRP2 and LHCGR were significantly associated (P<0.01) with the number of oocytes collected by ovum pickup, whereas the SNP in the mtDNA was not. In addition, we estimated an allelic substitution effect of 1.13±0.01 (P<0.01) oocytes for the SNP in the FGF8 gene. The results we report herein provide further evidence to support the hypothesis that genetic variability is an important component of the number of antral follicles in the bovine ovary.

VEGFA164_B mRNA Abundance Has a Positive Relationship to AMH, BCL2 and the Ratio of E2:A4 in Mural Granulosa Cells of Estrogen Active and Inactive Follicles Prior to Ovulation.

Previously, we reported differential expression of Vascular Endothelial Growth Factor A (VEGFA) isoforms in somatic cells from bovine females of differing fertility. Thus, we sought to determine how expression of VEGFA pro and antiangiogenic isoforms were regulated after synchronization protocols that stimulated normal or persistent follicle development. Furthermore, we examined relationships between VEGFA isoforms, steroid hormones and genes that regulate follicular development in mural granulosa cells and cumulus oocyte complexes (COC). Our hypothesis was that dominant estrogen active (EA) follicles would have greater mRNA abundance of VEGFA angiogenic isoforms and genes that stimulate follicle development; while estrogen inactive (EI) follicles would contain greater mRNA expression of VEGFA antiangiogenic isoforms. Thirty-five composite beef cows [75% MARC III (1/4 Angus, 1/4 Hereford, 1/4 Pinzgauer, 1/4 Red Poll) and 25% Red Angus] were treated with either 1) a modified Co-Synch protocol (CIDR) or 2) injected with prostaglandin and offered Melengesterol Acetate (MGA) for 14 days. Dominant follicle aspirations were performed 18, 36, and 60 hours after the last PGF2a injection utilizing transvaginal ultrasound guided ovum pickup. Follicular fluid steroid hormone concentrations were determined via RIA (E2 and P4) or ELISA (A4). Follicles were then classified as EA (E2:P4 > 1) or as EI (E2:P4 = 1). There was no effect of time on gene expression or steroid hormone levels, thus animals were grouped based on treatment and follicle E2-activity. CIDR and MGA EI follicles had a greater (P = 0.02) concentrations of progesterone compared to CIDR EA follicles. Although A4 concentration was similar among groups, CIDR EA follicles had greater E2:A4 ratio compared to EI follicles suggesting reduced efficiency in conversion of A4 to E2.To determine if the reduced E2:A4 ratio in EI follicles alters the mRNA abundance of VEGFA isoforms in the COC or mural granulosa cells quantitative, real-time RT-PCR was performed. There was no difference in VEGFA_164 gene expression among groups. However, abundance of VEGFA_164B mRNA was 7-fold and 2-fold greater (P < 0.01) in the mural granulosa cells of MGA EI and CIDR EI, respectively, compared to CIDR EA follicles. Furthermore, there was a trend (P = 0.06) for an increased ratio of VEGFA_164/ VEGFA_164B in mural granulosa of CIDR EA follicles. Follicles classified as MGA EI had greater (7-fold; P = 0.02) mRNA abundance of AMH compared to CIDR EA follicles in granulosa cells. Furthermore, BCL2 mRNA expression tended to be increased (P = 0.07) in MGA EI follicles. In contrast to the mural granulosa cells, VEGFA_164B mRNA abundance was not different in COC. In mural granulosa cells, there was a positive correlation between VEGFA_164B and BCL2; VEGFA_164B and AMH; VEGFA_164B and the ratio of E2:P4; and the ratio of VEGFA_164/VEGFA_164B and BCL2.Taken together these data suggest a tendency for increased levels of anti-apoptotic factors and reduced efficiency of conversion of A4 to E2 in granulosa cells of EI follicles. Furthermore, there is a positive correlation between the antiangiogenic VEGFA isoform and BCL2 and AMH which play a role in regulating follicle development. USDA is an equal opportunity provider and employer.

230 OVUM PICKUP - IN VITRO PRODUCTION EFFICIENCY FOR CONSERVATION OF THE FLAMENGA CATTLE BREED

The introduction of more specialised and productive dairy breeds into commercial herds in recent decades has caused a gradual loss of interest in dual-purpose cattle breeds, such as the Flamenga. Because of its importance to biodiversity, the high risk of loss of this small Brazilian herd, located in one geographic region in Southern Brazil, justifies the need for and efforts in genetic conservation of the Flamenga breed. The ovum pickup (OPU) procedure is a potential tool for such conservation purposes. Thus, the aim of this study was to evaluate the oocyte and embryo yield potential of the Flamenga breed, as compared with Holstein control counterparts, after in vivo oocyte retrieval followed by in vitro embryo production (IVP). Four Flamenga and 4 Holstein multiparous milking cows from the same herd were subjected to weekly ultrasound-guided OPU sections for 10 consecutive weeks. The number of visible follicles per ovary per female was recorded before the aspiration of ≥3-mm follicles. Retrieved cumulus–oocyte complexes (COC) were morphologically graded according to the method of (Leibfried and First 1979 J. Anim. Sci. 48, 76–86), and viable COC (Grades I, II, and III) from both breeds and from slaughterhouse ovaries (IVP controls) were used for the IVP of embryos, based on our established procedures (Vieira et al. 2002 Cryobiology 45, 91–94). Frozen semen from the same Flamenga breed bull was used in all groups. Quantitative (visible and aspirated follicles, collected COC), and qualitative (COC recovery efficiency, cleavage and blastocyst rates) data were compared between breeds and IVP controls (when pertinent) by Student’s t-test and chi-square test, respectively, for P &lt; 0.05. Results are presented in Table 1. The pools of visible and aspirated follicles were similar between breeds. However, the COC recovery efficiency was higher for Flamenga than for Holstein females. In vitro embryo development was also different between breeds, with cleavage rate being significantly lower for Holsteins than for Flamenga and IVP controls. Blastocyst rates also differed between treatments. Nonetheless, development to the blastocyst stage, when based on cleavage, was similar between Flamenga and Holstein embryos, but with both breeds being lower than the IVP controls. Oocyte quality was likely the cause of such differences in embryo developmental potential between groups, with Flamenga females yielding more oocytes and IVP embryos than Holstein cows. In summary, the use of OPU-IVP procedures was demonstrated to be suitable for the preservation of the Flamenga breed in Southern Brazil. Table 1.Follicular pool and oocyte and embryo yield between Flamenga and Holstein cows following ovum pickup–in vitro production (IVP) procedures This study was supported by CAPES and CNPq/Brazil.