Multiple Ovulation Research Articles

Towards Improving the Outcomes of Multiple Ovulation and Embryo Transfer in Sheep, with Particular Focus on Donor Superovulation.

Considerable improvements in sheep multiple ovulation and embryo transfer (MOET)protocols have been made; however, unlike for cattle, MOET is poorly developed in sheep, and thus has not been broadly applicable as a routine procedure. The tightly folded nature of the ewe cervix, the inconsistent ovarian response to various superovulatory treatments, and the requirement of labor to handle animals, particularly during large-scale production, has limited the implementation of successful MOET in sheep. Moreover, several extrinsic factors (e.g., sources, the purity of gonadotrophins and their administration) and intrinsic factors (e.g., breed, age, nutrition, reproductive status) severely limit the practicability of MOET in sheep and other domestic animals. In this review, we summarize the effects of different superovulatory protocols, and their respective ovarian responses, in terms of ovulation rate, and embryo recovery and transfer. Furthermore, various strategies, such as inhibin immunization, conventional superovulation protocols, and melatonin implants for improving the ovarian response, are discussed in detail. Other reproductive techniques and their relative advantages and disadvantages, such as artificial insemination (AI), and donor embryo recovery and transfer to the recipient through different procedures, which must be taken into consideration for achieving satisfactory results during any MOET program in sheep, are also summarized in this article.

Flower Bud Differentiation, Mega-micro Spore Production, and Male-female Gametophyte Development in Camellia weiningensis

Camellia weiningensis is a typical woody edible oil tree species in the northwest alpine area of Guizhou Province, China, but its embryological development is not fully elucidated. Here, we assessed flower bud differentiation, microsporogenesis, and male-female gametophyte development in this species. We performed cytological observations of flower bud development in C. weiningensis through conventional paraffin sectioning, scanning electron microscopy, and stereomicroscopy to establish the corresponding relationships between the external morphology and internal structure. The flowers were hermaphroditic and exhibited a short flower bud differentiation time. Although pistil development occurred later than stamen development, both organs matured synchronously before flowering. The anther contained four sacs that exhibited a butterfly shape in transverse sections. The anther wall comprised the epidermis, anther chamber inner wall, two middle layers, and a glandular tapetum (from outside to inside). Microspore mother cells formed a tetrahedral tetrad through meiosis, mature pollen was two-celled with three germination pores, and the ovary comprised three to five chambers (three chambers predominated). Multiple ovules were invertedly attached to the axial placentation and exhibited double integuments and a thin nucellus. The embryo sac exhibited Allium-type development, and the mature embryo sac was seven-celled and eight-nucleated. In C. weiningensis, embryonic development does not exhibit abnormalities, and stamen development occurs earlier than pistil development. During flower bud development, the inner development process of male and female cells can be judged according to their external morphological characteristics. Our results may provide a theoretical basis for regulating flowering in and the cross-breeding of C. weiningensis.

Relationship between the concentration of anti-Müllerian hormone and antral follicle count in pasture-managed Holstein cows in the high tropics.

We evaluated the relationship between plasma levels of anti-Müllerian hormone (AMH) and the number of antral follicles at the restart of the follicular wave in crossbred Holstein cows reared under extensive grazing systems over 2500 m above sea level. The study included 140 cows from 15 farms that were in average at the 75.3 ± 2.10 d post partum. Animals were synchronized according to the following regime: day 0 = intravaginal progesterone releasing device (IPD) + estradiol benzoate (EB); day 7: withdrawal of IPD + prostaglandin; and day 8: EB, for restart of the follicular wave on day 11. On this day 11, antral follicle counts (AFCs) were made by transrectal ultrasound, and a plasma sample was taken for the determination of AMH. The mean AMH plasma level was 0.06 ± 0.03 ng/ml and the mean AFC was 17.26 ± 0.38 follicles. A strong positive linear correlation was found between these two variables (r = 0.783, r = 0.613, P < 0.0001). Cows were categorized according to AMH concentration as high (>0.09 ng/ml), intermediate (0.09-0.05 ng/ml) or low (<0.05 ng/ml). Cows with high AMH presented a higher AFC (25.0 ± 2.21 follicles) than those with low AMH (14.08 ± 2.68 follicles; P < 0.001. Our results suggest that the cut-off value of AMH = 0.09 ng/ml may be useful for selecting donors in multiple ovulation embryo transfer programs involving cows with these characteristics. Our data further suggest that AMH plasma concentration correlates with AFC and can be used as an endocrine biomarker of the number of antral follicles present at a given moment of the estrous cycle in crossbred Holstein cows raised at altitudes above 2500 m.

MOET Efficiency in a Spanish Herd of Japanese Black Heifers and Analysis of Environmental and Metabolic Determinants.

Simple SummaryEmbryo transfer procedures have been widely implemented in bovine all around the world. These techniques help to accelerate the increase in the genetic merit and to keep the level of inbreeding under control, which is especially important in breeds, such as Japanese Black cattle, in areas outside of Japan, where there are scarce individuals and few herds. Our study describes an adequate embryo productivity of Japanese Black heifers under Spanish management and environmental conditions, like that which has been previously reported, demonstrating the adequate adaptation capacity of these animals. No effect on embryo production or quality was found due to plasma metabolic parameters of the donors, suggesting an optimal nutritional and body conditioning of the donors’ herd. Pregnant recipients after embryo transfer showed significantly higher levels of cholesterol-related parameters, glucose, and urea, which may be related to higher availability of nutrients for the future pregnancy. Heat stress at embryo transfer negatively impacted conception rates as expected, while larger parity and ET number corresponded to numerically higher conception rates.Multiple ovulation and embryo transfer (MOET) systems have been intensively implemented in Japanese Black cattle in Japan and to create Japanese Black herds out of these areas. Environmental conditions influence MOET efficiency. Thus, we describe results of 137 in vivo, non-surgical embryo flushings performed between 2016–2020, in a full-blood Japanese Black herd kept in Spain and the possible effects of heat, year, bull, donor genetic value, and metabolic condition. Additionally, 687 embryo transfers were studied for conception rate (CR) and recipient related factors. A total of 71.3% of viable embryos (724/1015) were obtained (5.3 ± 4.34/flushing). Donor metabolites did not affect embryo production (p > 0.1), although metabolite differences were observed over the years, and by flushing order, probably related to the donor age. CR was not affected by embryo type (fresh vs. frozen), recipient breed, and whether suckling or not suckling (p > 0.1). CR decreased significantly with heat (44.3 vs. 49.2%; (p = 0.042)) and numerically increased with recipient parity and ET-number. Pregnant recipients showed significantly higher levels of cholesterol-related metabolites, glucose, and urea (p < 0.05). Therefore, adequate MOET efficiency can be achieved under these conditions, and heat stress should be strongly avoided during Japanese Black embryo transfers. Moreover, recipients’ metabolites are important to achieve pregnancy, being probably related to better nutrient availability during pregnancy.

Effects of Extra-Long-Acting Recombinant Bovine FSH (bscrFSH) on Cattle Superovulation.

Simple SummaryWhile the clinical effectiveness of medium-acting bovine follicle stimulating hormone (bFSH) is well-established for superovulation (SOV) protocols in cattle, the use of long-acting bovine recombinant follicle stimulating hormone (brFSH) remains the golden target. The application of a bFSH with prolonged half-life could improve substantially the results obtained regarding the ovarian superstimulation response and the quality and quantity of the embryos produced. Moreover, the management schemes may be improved by reducing the number of FSH applications needed during the SOV protocol. The development of a new extra-long-acting bovine single-chain recombinant follicle stimulating hormone (bscrFSH) provides interesting advantages to the SOV protocols as the present study shows, by improving the results obtained compared to the use of the conventional follicle stimulating hormone from purified pig pituitary extract (NIH-FSH-p).Over the last few years, several commercial FSH products have been developed for cattle superovulation (SOV) purposes in Multiple Ovulation and Embryo Transfer (MOET) programs. The SOV response is highly variable among individuals and remains one of the main limiting factors in obtaining a profitable number of transferable embryos. In this study, follicle stimulating hormone (FSH) from different origins was included in two SOV protocols, (a) FSH from purified pig pituitary extract (NIH-FSH-p; two doses/day, 12 h apart, four consecutive days); and (b) extra-long-acting bovine recombinant FSH (bscrFSH; a single dose/day, four consecutive days), to test the effects of bscrFSH on the ovarian response, hormone profile levels, in vivo embryo production and the pluripotency gene expression of the obtained embryos. A total of 68 healthy primiparous red Angus cows (Bos taurus) were randomly distributed into two experimental groups (n = 34 each). Blood sample collection for progesterone (P4) and cortisol (C) level determination was performed together with ultrasonographic assessment for ovarian size, follicles (FL) and corpora lutea (CL) quantification in each SOV protocol (Day 0, 4, 8, and 15). Moreover, FSH profiles were monitorised throughout both protocols (Day 0, 4, 5, 6, 7, 8, 9, 10, and 15). In vivo embryo quantity and quality (total structures, morulae, blastocysts, viable, degenerated and blocked embryos) were recorded in each SOV protocol. Finally, embryo quality in both protocols was assessed by the analysis of the expression level of crucial genes for early embryo development (OCT4, IFNt, CDX2, BCL2, and BAX). P4 and cortisol concentration peaks in both SOV protocols were obtained on Day 15 and Day 8, respectively, which were statistically different compared to the other time-points (p < 0.05). Ovarian dimensions increased from Day 0 to Day 15 irrespective of the SOV protocol considered (p < 0.05). Significant changes in CL number were observed over time till Day 15 irrespective of the SOV protocol applied (p < 0.05), being non- significantly different between SOV protocols within each time-point (p > 0.05). The number of CL was higher on Day 15 in the bscrFSH group compared to the NIH-FSH-p group (p < 0.05). The number of embryonic structures recovered was higher in the bscrFSH group (p = 0.025), probably as a result of a tendency towards a greater number of follicles developed compared to the NIH-FSH-p group. IFNt and BAX were overexpressed in embryos from the bscrFSH group (p < 0.05), with a fold change of 16 and 1.3, respectively. However, no statistical differences were detected regarding the OCT4, CDX2, BCL2, and BCL2/BAX expression ratio (p > 0.05). In conclusion, including bscrFSH in SOV protocols could be an important alternative by reducing the number of applications and offering an improved ovarian response together with better embryo quality and superior performance in embryo production compared to NIH-FSH-p SOV protocols.

ИТОГИ И ПЕРСПЕКТИВЫ ПРИМЕНЕНИЯ ВСПОМОГАТЕЛЬНЫХ РЕПРОДУКТИВНЫХ ТЕХНОЛОГИЙ В ВОСПРОИЗВОДСТВЕ МЕЛКИХ ЖВАЧНЫХ

The article discusses the most common ART (artificial insemination, cryopreservation of sperm and creation of storage facilities, induction and synchronization of sexual reproduction), as well as ART used in rare cases (technology of superovulation induction and embryo transfer, in-vitro embryo production, embryo cryopreservation, transgenesis). The author presents the results of own long-term research on the development of biotechnological methods and methods of increasing the reproductive qualities of sheep and goats. The reasons for the rare use of multiple ovulation and embryotransplantation programs (MOET) in domestic sheep and goat breeding practice are given. Recommendations on using MOET to preserve the gene pool and to increase the efficiency of using genetic resources of high-value animals, increasing their participation in the sheep and goat reproduction process, are given.

Multiple ovulations between ovarian stimulation treatment and primary ovarian insufficiency

The release of several oocytes during the same menstrual cycle can occur both physiologically and iatrogenic, following ovarian stimulation treatments. Ovarian induction can be achieved either by mild stimulation, in the case of anovulatory or dysovulatory patients, or by controlled ovarian hyperstimulation, to obtain a large number of follicles in the In Vitro Fertilization procedure. At this time, the ideal ovarian stimulation treatment is to suppress the pituitary release of gonadotrophin-releasing hormone (GnRH), a co-opted regimen with high doses of exogenous follicle-stimulating hormone (FSH). The physiological process of multiple ovulations can lead to superfertilization (SF). SF is a biological process by which two eggs are fertilized successively, at intervals of a few days apart, both eggs coming from the same ovulation. Among the factors that can lead to superfertilization are mentioned: family history of dizygotic pregnancies (DZ), increased parity, advanced age, high stature of women, a high weight index, the use of oral contraceptives and folic acid. The premenopausal period is characterized by elevated levels of FSH and a low ovarian reserve, both conditions contributing to the development of multiple follicular and DZ pregnancies.

Occurrence of premature regression of corpus luteum in MOET programs in Dorper ewes under subtropical climate

Premature regression of corpus luteum (PRCL) is a functional alteration that occurs mainly in superovulated females hampering the widespread use of multiple ovulation and embryo transfer (MOET). This study evaluated the association of donor age (3–9 years old), body condition score (2 to 4.5), number of MOET (1 to 5) repetitions and season (breeding, transition, or non-breeding) with the occurrence of PRCL in ewes. Multiparous Dorper ewes (n = 44) were subjected to MOET during four consecutive years, with the same superovulatory treatment. Before embryo collection, ovaries were laparoscopically performed to assess the corpora lutea (CL). Overall, the total PRCL occurrence was 24.7% (26/105) and it had no association with the studied factors. Percentages of ewes that presented PRCL once or twice were 45.5% (20/44) and 6.8% (3/44), respectively, while the percentage of ewes that had no PRCL was 47.7% (21/44). Proportions of PRCL were similar (P > 0.05) among breeding (21%), transition (31%), and non-breeding (24%) season; and also (P > 0.05) in lower/thin (20%), average/good (24%), and higher/fat (29%) females. In conclusion, although this phenomenon had no association to the factors studied, the occurrence of PRCL affects ∼25% of ewes subjected to MOET procedures and future alternatives to circumvent this obstacle need to be developed.

Dairy cattle farmers' preferences for different breeding tools

Breeding technologies play a significant role in improving dairy cattle production. Scientifically proven tools for improved management and genetic gain in dairy herds, such as sexed semen, beef semen, genomic testing, dairy crossbreeding, and multiple ovulation embryo transfer (MOET), are readily available to dairy farmers. However, despite good accessibility, decreasing costs, and continuous development of these tools, their use in Sweden is limited. This study investigated Swedish dairy farmers' preferences for breeding tools through a survey including a discrete choice experiment. The survey was distributed online to 1 521 Swedish farmers and by an open link published through a farming magazine. In total, the study included 204 completed responses. The discrete choice experiment consisted of 10 questions with two alternative combinations, which gave 48 combinations in total. Utility values and part-worth values were computed using a conditional logit model based on the responses in the discrete choice experiment for nine groups of respondents: one group with all respondents, two groups based on respondents using dairy crossbreeding or not within the past 12 months, two based on herd size, two based on respondent age, and two based on whether respondents had used breeding advisory services or not. The strongest preferences in all groups were for using sexed semen and beef semen. Genomic testing was also significantly preferred by all groups of respondents. Except in large herds, MOET on own animals was significantly and relatively strongly disfavoured by all groups. Buying embryos had no significant utility value to any group. Dairy crossbreeding had low and insignificant utility values in the group of all respondents, but it was strongly favoured by the group that had used dairy crossbreeding within the past 12 months, and it was disfavoured by the group that had not. Part-worth values of combined breeding tools showed that combinations of sexed and beef semen, alone or with genomic testing without dairy crossbreeding, were the most preferred tools. Compared with the most common combinations of breeding tools used in the past 12 months, the part-worth values indicated that Swedish dairy farmers may prefer to use breeding tools more than they do today. Statements on the different breeding tools indicated that the respondents agreed with the benefits attributed to the breeding tools, but these benefits may not be worth the cost of genomic testing and the time consumption of MOET. These valuable insights can be used for further development of breeding tools.

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.

Factors influencing embryo quantity and quality in donor cows from Latvian Blue, Latvian Brown, and Danish Red breeds at risk in Latvia

The aim of this study was to find out what general factors influenced embryo quantity and quality in the framework of multiple ovulation and embryo transfer done in Latvia 2018–2020. The gene-pool cows from the cattle breeds Latvian Blue, Latvian Brown, and Danish Red (n = 30) were selected. Such factors as the donor`s blood biochemical and hematological parameters value, ovarian steroid hormones level, milk somatic cell count (SCC), parity, and environmental temperature were analyzed using a negative-binomial regression method. The variables significant at a liberal alpha level of 0.20 in univariable regression models were included in two multivariable models. The first model showed that the number of embryos (nE) was associated (P < 0.01) with a higher progesterone (P4) level and blood absolute count of eosinophils (Eo) on the first day of multiple ovulation (MO) treatment, with a lower SCC and average daily air temperature in the month preceding embryo collection, and lower parity. It can be predicted according to the nE model, if P4 level or Eo absolute count is increasing by the interquartile range (IQR), and provided that the other associated parameters remain constant, the nE will be higher by 2.9 and 4.4 embryos, respectively. If SCC or temperature is increasing by the IQR, the nE will be lower by 4.7 and 15.2 embryos, respectively. Additionally, the nE model predicted 13.3 more embryos in primiparous than in multiparous donors. The second model showed that the number of transferable embryos (nTE) was associated (P < 0.05) with a higher Eo and serum sodium (Na+) concentration on the first day of MO treatment, and with a lower SCC and average daily air temperature in the month preceding embryo collection. It can be predicted according to the nTE model, if Na+ level or Eo absolute count is increasing by the IQR, the nTE will be higher by 3.7 and 2.4 embryos, respectively. If SCC or temperature is increasing by the IQR, the nTE will be lower by 3.3 and 6.4 embryos, respectively.

Personalized medicine for GnRH antagonist protocol in in vitro fertilization procedure using modeling and optimal control

Infertility is an inability of those of reproductive age to become or remain pregnant within five years of exposure to pregnancy WHO. Since its inception in 1978, In vitro fertilization (IVF) has become the most resorted protocol for treating infertility. This article is focused on the first stage of IVF namely, superovulation. Superovulation is the drug-induced ovarian stimulation process to facilitate multiple ovulations per menstrual cycle. Thus, successful superovulation is the foundation for a successful IVF cycle. Despite the existence of general guidelines for dosage prescription, patient customized dosage protocols do not exist, and complications, such as overstimulation, do occur. To solve the limitations of the existing empirical system, a mathematical algorithm is developed to provide a customized model of superovulation owing to the size distribution of eggs (follicles/ oocytes) obtained per cycle as a function of the chemical interactions of the drugs used and the patient conditions during the cycle, to serve as a baseline for forecasting the outcome. A personalized medicine approach was previously presented based on mathematical modeling and optimal control for the agonist Long Lupron protocol of IVF(Nisal et al., 2020). This article is an extension of that work and describes the theory, modeling, and optimal control strategy to improve outcomes of IVF treatment for the antagonist Ganirelix (GnRH) protocol used in clinical practice. The validation of the procedure is performed using clinical data from the patients previously undergone IVF cycles. The data was available for 13 patients and customized patient-specific model parameters were obtained from the data. The model was used to predict follicle size distribution for all 13 patients for the remaining days of the cycle. It was found that the personalized models showed a strong fit with the clinically observed follicle size distribution (FSD). Then, the optimal control method was used for each individual patient to predict optimized drug dosage protocols using the customized patient specific parameters. The validity of modeling and optimization approach is corroborated by the results and show the efficacy of the optimized drug dosage protocols for each patient.

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.

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.

Epigenomic and transcriptomic analyses reveal early activation of the HPG axis in in vitro-produced male dairy calves.

In cattle, several calves born after IVP ("in vitro" embryo production) present similar birthweight to those generated after MOET (multiple ovulation and embryo transfer). However, the underlying molecular patterns in organs involved in the developmental process are unknown and could indicate physiological programming. The objectives of this study were: (1) to compare epigenomic and transcriptomic modifications in the hypothalamus, pituitary, gonadal and adrenal organs between 3months old ovum pick-up-IVP and MOET male calves (n=4 per group) and (2) to use blood epigenomic data to proxy methylation of the inner organs. Extracted gDNA and RNA were sequenced through whole-genome bisulfite sequencing and RNA sequencing, respectively. Next, bioinformatic analyses determined differentially methylated cytosines (DMC) and differentially expressed genes (DEG) (FDR<0.05) in IVP versus MOET samples and the KEGG pathways that were overrepresented by genes associated with DMC or DEG (FDR<0.1). Pathways related to hypothalamus, pituitary, gonadal (HPG) axis activation (GnRH secretion in the hypothalamus, GnRH signaling in the pituitary, and steroidogenesis in the testicle) were enriched in IVP calves. Modeling the effect of the methylation levels and the group on the expression of all the genes involved in these pathways confirmed their upregulation in HPG organs in IVP calves. The application of the DIABLO method allowed the identification of 15 epigenetic and five transcriptomic biomarkers, which were able to predict the embryo origin using the epigenomic data from the blood. In conclusion, the use of an integrated epigenomic-transcriptomic approach suggested an early activation of the HPG axis in male IVP calves compared to MOET counterparts, and the identification of potential biomarkers allowed the use of blood samples to proxy methylation levels of the relevant internal organs.

Case report of partial empty sella syndrome: A challenging cocktail for the anesthesiologist

Background: Eventhough panhypopituitarism is a rare endocrine system disease, the clinical manifestation can vary from subclinical to life threatening myxoedema coma. It could be due to either primary pituitary dysfunction/stalk dysfunction/hypothalamic dysfunction.A 32 year old young female who presented with bilateral hip joint pain was diagnosed to have bilateral avascular necrosis of femoral head. On ellicting a detailed clinical history, she underwent treatment for infertility for 7 long years which comprised of multiple ovulation induction agents and finally conceived following in utero insemination. Although the gestational and peripartum period were uneventful, she had agalactorrhea, a warning sign of impending hormonal dysfunction which didn’t seek much attention. Five years down the lane, on evaluation for dyslipidemia, she was found to have hypothyroidism, hypocalcemia, low serum calcitriol levels. A detailed brain imaging studies showed partially emptysella with small remnant pituitary tissue and was started on treatment as a case of panhypopituitarism.

A retrospective study investigating the association of parity, breed, calving month and year, and previous parity milk yield and calving interval with twin births in US dairy cows

Twinning costs the dairy industry an estimated $96 million each year. Twin pregnancy occurrence in high-producing dairy cows is primarily a result of multiple ovulations associated with low circulating concentrations of progesterone due to high milk production. The present retrospective observational study aimed to identify associations between (1) previous parity milk yield and subsequent twin birth prevalence, (2) twin birth with same parity milk production and calving interval (CInt), and (3) twin birth and the subsequent twin calving. The final data set included almost 2.9 million US dairy calving and production records between 2001 and 2020. Variables considered were parity, breed, milk production, CInt, calving month, and year. Logistic and linear regression modeling were used to assess the effects of predictors on outcomes. Herd within state was used as a random effect for all regression models. Twin birth probability increased for cows with increased previous parity milk yield independent of breed or parity. Third and greater parity (3+) compared with second parity (2) and all breeds compared with Jerseys were associated with greater twin probability. Calving between April and September that corresponded to conceiving in July through December was associated with greater twin birth probability. Twin births were associated with decreased milk production following the birth event in Holsteins and parity 2 cows and in the calving months between June and September. Surprisingly, twin births in parity 3+ cows were associated with an increased 305-d milk yield. Cows that had a twin birth were more likely to calve twins in the subsequent parity and had a greater risk of having a CInt between 413 and 600 d. The hazard to subsequent calving after single births was greater compared with twin births. These data can be instrumental in guiding research focus on reducing twinning in lactating dairy cows.

Thermal Mechanisms Preventing or Favoring Multiple Ovulations in Dairy Cattle.

Simple SummaryWhile cows are usually monovular, the incidence of dizygous twin births has recently increased considerably alongside increasing milk production. Genetic progress and improvements in nutrition and management practices have led to a continuous increase in milk yield and thus also to multiple ovulations and twin pregnancies. Twin pregnancies are undesirable as they seriously compromise the welfare of the cow and herd economy. A better understanding of the processes associated with multiple ovulations should help to reduce rates of twinning. During the stages of the sexual cycle, temperature gradients are established within the ovary and throughout the genital tract. Pre-ovulatory local cooling of the reproductive system favors male and female gamete maturation and subsequent fertilization. In fact, thermal mechanisms may prevent or favor multiple ovulations and thus twinning. The purpose of this review was to update this topic.While cows are predominantly monovular, over the past 30 years the incidence of multiple ovulations and thus twinning has increased considerably alongside milk production. Multiple pregnancies are not desirable as they negatively affect the health of cows and the herd economy. Although causal mechanisms associated with multiple ovulations have been extensively revised, the process of multiple ovulations is not well understood. Recent studies on the thermal biology of the reproductive system have shown how thermal mechanisms may prevent or favor multiple ovulations. This review focuses on this relationship between thermal dynamics and multiple pregnancies. Cooling of the pre-ovulatory follicle is able to regulate ovulation. In effect, pre-ovulatory local cooling of the female reproductive system favors male and female gamete maturation and promotes fertilization. Thermal stress is proposed here as a model of stress. Periods of high ambient temperature affect the processes of pre-ovulatory follicular cooling and multiple ovulations. While the ratio between unilateral and bilateral multiple pregnancies is normally close to one, under heat stress conditions, this ratio may be 1.4 favoring unilateral multiple pregnancies. A ratio approaching unity is here proposed as an indicator of cow wellbeing.

Multiple ovulation and embryo transfer during the breeding season in Angora goats: A comparison of fresh and vitrified-thawed embryo transfer.

This study aimed to assess the superovulation response and pregnancy rates of fresh and vitrified-thawed embryos after transfer in Angora goats with comparing transfer at the beginning (BS) and end (ES) of the breeding season. Nine Angora goats were used as donors in both periods. Donor goats were synchronized and superovulated with the FSH and mated with five fertile bucks. At 156 hr following mating, embryos were collected surgically. Recipient Angora goats were divided into two groups at the beginning (fresh, n = 15; vitrified-thawed, n = 15) and end (fresh, n = 8; vitrified-thawed, n = 8) of the breeding season. Fresh or vitrified-thawed grade I embryos (early blastocyst/blastocyst) were transferred surgically to synchronized recipients. On the 30th, 60th and 90th day of transfer, goats were examined by ultrasonography. The number of corpora lutea (CL), total oocyte/embryo and transferable embryos obtained in BS was found to be statistically higher than ES. On the 30th day of transfer, pregnancy rates were 73.30% and 75.00% in the fresh transfer groups in both BS and ES periods; while, rates of 20.00% and 37.50% were found in the vitrified-thawed group, respectively. The embryo survival rates of fresh transfers were 55.55% and 31.25% at BS and ES, respectively. The number of CL, total oocyte/embryo and transferable embryos in the BS was higher than ES following superovulation. Also, fresh embryo transfer can be successfully carried out during the breeding season in Angora goats. Moreover, although pregnancies were obtained following the transfer of vitrified-thawed embryos, they did not sustain on the 60th and 90th days. So, further studies are needed for the vitrified-thawed embryos.

Technological approaches to the problem of double ovulation and twin pregnancies in mares

The aim of the work was to determine and justify the correct technological approach in cases of double ovulation and twin pregnancies in mares. The frequency of single and double ovulations (n=2207), the level of twin pregnancies and the results of manual reduction of excess embryo in mares were studied. The average level of double ovulations was 9.5 % per year, and it had no significant difference in mares of different breeds (Russian Trotter, French Trotter, Standardbred and others). The number of poliovulations in February-March (3.2–4.9 %) and August (5.1 %) was significantly (p <0.05) lower than it was in April-July (9.9–11.3 %). The number of multiple ovulations gradually decreased from young (4–8 yo) to old (>14 yo) mares (11.9 – 5.1 %) (p <0.05). The third part of all mares (35.8 %) with poliovulations repeated it during one season, and after 1–3 successive breeding seasons. The mating of mares with double ovulation resulted in 35.0 % of twin embryos and 28.1 % of single embryos. The success rate of manual reduction of the second embryo on days 12–14 was 85.3 %.