Fetal sex alters maternal anti-Mullerian hormone during pregnancy in cattle.

Anti-Mullerian hormone (AMH) is expressed by both male and female fetuses during mammalian development, with males expressing AMH earlier and at significantly higher concentration. The aim of the current study was to explore the potential impact of pregnancy and fetal sex on maternal AMH and to determine if plasma (Pl) AMH or placenta intercotyledonary membrane and cotyledonary AMH receptor 2 (AMHR2) mRNA expression differ in pregnant cows carrying male vs. female fetuses. AMH levels in blood were measured using a bovine optimized ELISA kit. Cows pregnant with a male fetus were observed to have a significantly greater difference in Pl AMH between day 35 and 135 of gestation. Average fetal AMH level between 54 and 220days of gestation was also observed to be significantly higher in male vs. female fetuses. Intercotyledonary membranes and cotyledons were found to express AMHR2 between days 38 and 80 of gestation at similar levels in both fetal sexes. These findings support the hypothesis that fetal sex alters maternal Pl AMH during pregnancy in cattle.

Increasing of blastocyst rate and gene expression in co-culture of bovine embryos with adult adipose tissue-derived mesenchymal stem cells.

PURPOSE: Despite advances in the composition of defined embryo culture media, co-culture with somatic cells is still used for bovine in vitro embryo production (IVEP) in many laboratories worldwide. Granulosa cells are most often used for this purpose, although recent work suggests that co-culture with stem cells of adult or embryonic origin or their derived biomaterials may improve mouse, cattle, and pig embryo development. MATERIALS AND METHODS: In experiment 1, in vitro produced bovine embryos were co-cultured in the presence of two concentrations of bovine adipose tissue-derived mesenchymal cells (b-ATMSCs; 103 and 104 cells/mL), in b-ATMSC preconditioned medium (SOF-Cond), or SOF alone (control). In experiment 2, co-culture with 104 b-ATMSCs/mL was compared to the traditional granulosa cell co-culture system (Gran). RESULTS: In experiment 1, co-culture with 104 b-ATMSCs/mL improved blastocyst rates in comparison to conditioned and control media (p < 0.05). Despite that it did not show difference with 103 b-ATMSCs/mL (p = 0.051), group 104 b-ATMSCs/mL yielded higher results of blastocyst production. In experiment 2, when compared to group Gran, co-culture with 104 b-ATMSCs/mL improved not only blastocyst rates but also quality as assessed by increased total cell numbers and mRNA expression levels for POU5F1 and G6PDH (p < 0.05). CONCLUSIONS: Co-culture of bovine embryos with b-ATMSCs was more beneficial than the traditional co-culture system with granulosa cells. We speculate that the microenvironmental modulatory potential of MSCs, by means of soluble substances and exosome secretions, could be responsible for the positive effects observed. Further experiments must be done to evaluate if this beneficial effect in vitro also translates to an increase in offspring following embryo transfer. Moreover, this study provides an interesting platform to study the basic requirements during preimplantation embryo development, which, in turn, may aid the improvement of embryo culture protocols in bovine and other species.

Systemic and local anti-Mullerian hormone reflects differences in the reproduction potential of Zebu and European type cattle.

This study was conducted to evaluate plasma anti-Mullerian hormone (Pl AMH), follicular fluid AMH (FF AMH) and granulosa cell AMH transcript (GC AMH) levels and their relationships with reproductive parameters in two cattle subspecies, Bos taurus indicus (Zebu), and Bos taurus taurus (European type cattle). Two-dimensional ultrasound examination and serum collection were performed on Zebu, European type and crossbreed cows to determine antral follicle count (AFC), ovary diameter (OD) and Pl AMH concentration. Slaughterhouse ovaries for Zebu and European type cattle were collected to determine FF AMH concentrations, GC AMH RNA levels, AFC, oocyte number, cleavage and blastocyst rate. Additionally GC AMH receptor 2 (AMHR2) RNA level was measured for European type cattle. Relationship between AMH and reproductive parameters was found to be significantly greater in Zebu compared to European cattle. Average Pl AMH mean ± SE for Zebu and European cattle was 0.77 ± 0.09 and 0.33 ± 0.24 ng/ml respectively (p = 0.01), whereas average antral FF AMH mean ± SE for Zebu and European cattle was 4934.3 ± 568.5 and 2977.9 ± 214.1 ng/ml respectively (p < 0.05). This is the first published report of FF and GC AMH in Zebu cattle. Levels of GC AMHR2 RNA in European cattle were correlated to oocyte number (p = 0.01). Crossbred animals were found more similar to their maternal Zebu counterparts with respect to their Pl AMH to AFC and OD relationships. These results demonstrate that AMH reflects differences between reproduction potential of the two cattle subspecies therefore can potentially be used as a reproductive marker. Furthermore these results reinforce the importance of separately considering the genetic backgrounds of animals when collecting or interpreting bovine AMH data for reproductive performance.

Addition of L-arginine to the fertilization medium enhances subsequent bovine embryo development rates.

Although L-Arginine (ARG) has been reported as a promising bovine sperm capacitation agent, its effects on embryo development are still poorly understood. Herein, we compared the effects of ARG and/or heparin (HEP) addition to the fertilization medium for bovine oocytes on sperm capacitation and embryo development. We chose 10 mM ARG based on blastocyst development rates in a titration experiment. Addition of ARG and/or HEP to the fertilization medium resulted in similar rates of blastocyst development (P > 0.05). However, when ARG, but not HEP, was combined with a nitric oxide (NO) synthase inhibitor (N-Nitro-L-ARG-methyl ester, 10 mM) blastocyst development was decreased (P < 0.05). To assess the effects on capacitation, bovine sperm were incubated for 0, 3, and 6 hours in fertilization medium containing ARG and/or HEP and/or N-Nitro-L-ARG-methyl esterand acrosomal exocytosis rates were evaluated using fluorescein isothiocyanate conjugated Pisum sativum lectin (FITC-PSA) staining and flow cytometry. With HEP, acrosomal exocytosis rates were highest by 3 hours of incubation; however, by 6 hours, rates were similar for HEP and/or ARG (P > 0.05) and higher than those in control media (P < 0.05). Although both ARG and HEP increased sperm NO production (P < 0.05), combination with L-NAME only precluded acrosomal exocytosis when ARG added alone in the medium (P > 0.05). These results suggest that although both ARG and HEP supported sperm capacitation, only the effects of the former were driven via NO production. Moreover, ARG was also as effective as HEP at improving blastocyst development rates. Therefore, ARG may be used as a low-cost alternative sperm capacitation agent for bovine in vitro embryo production.

Effect of dexamethasone on development of in vitro-produced bovine embryos.

Studies in somatic cells have shown that glucocorticoids such as dexamethasone (DEX) may trigger or prevent apoptosis depending on the cell type in culture. Because the dysregulation of apoptosis may lower in vitro embryo production efficiency, we sought to investigate the effects of supplementing IVC medium with DEX (0.1 µg/mL) on embryo morphology, development kinetics, and apoptosis rates of in vitro-produced bovine preimplantation embryos. Embryo morphology was graded on Day 7, and development rates were assessed on Days 4 and 7 of IVC. Apoptosis was evaluated via annexin/propidium iodide staining under fluorescence microscopy where a cell labeled with annexin, propidium iodide, or both would be considered apoptotic. An embryo was counted in the apoptosis rates, if it displayed at least one such labeled cell. Although DEX supplementation did not reduce apoptosis rates, it had a positive impact on developmental kinetics and cell number both on Days 4 and 7 of embryo culture. Presumably, such effect resulted from increased cell proliferation rather than a direct inhibition of apoptosis. Further studies may evaluate the mechanisms by which glucocorticoids may affect embryo development, as DEX supplementation could become a tool to improve in vitro embryo yield in mammalian species.