Scrotal thermoregulation and sequential sperm abnormalities in buffalo bulls (Bubalus bubalis) under short-term heat stress.

Heat stress reduces the reproductive capacity of bulls raised in tropical climate. However, the reestablishment of scrotal thermoregulation and the dynamics of sperm defects emergence after stress are not completely known in buffaloes. Thus, the study aimed to evaluate the effect of short-term heat stress over scrotal thermoregulation and sperm attributes, relating them to spermatogenesis stages. Five buffalo bulls went through scrotal insulation during 48 h (from day 0 to day 2). Semen samples were collected every 7 days (from day -7 to day 49) and analyzed about the progressive motility, viability, and sperm morphology. Heat stress significantly destabilized scrotal thermoregulation (P < 0.001). Scrotal temperature was from 4.2 to 6.3 °C lower than the core body temperature, except on insulation days (P < 0.001), and returned to the basal condition five days after the removal of the stressing stimulus. More significant deleterious effects were observed in sperm morphology than in cell concentration, motility, and viability. The chronology of morphologic defects expression demonstrated tail defects (days 7-14), cytoplasmic droplets (days 14-28), and head defects (day 28), returning to pre-insulation condition 35 days after the thermal challenge. Thus, hyperthermia harmed more intensely spermatozoa in epididymal transit, elongated spermatids, and secondary spermatocytes. It is concluded that water buffalo bulls present a peculiar manifestation of sperm morphology after short-term stress, indicating an important difference related to the bovine species. Therefore, during the andrological evaluation of buffalo bulls, it is necessary to avoid the allometric extrapolation between these species.

Sperm chromatin protamination influences embryo development in unsexed and sexed bull semen.

Sex selection through sperm sorting offers advantages in regards selection pressure in high-producing livestock. However, the sex-sorting process results in sperm membrane and DNA damage that ultimately decrease fertility. We hypothesized that given the role of protamines in DNA packaging, protamine deficiency could account, at least partially, for the DNA damage observed following sperm sex sorting. To test this, we compared protamine status between unsexed and sexed spermatozoa from two bulls using the fluorochrome chromomycin A3 (CMA3) and flow cytometry. Then, we assessed embryo development following in vitro fertilization (IVF) using the same sperm treatments. Overall, sperm protamination was not different between sexed and unsexed semen. However, one of the two bulls displayed higher rates of protamine deficiency for both unsexed and sexed semen (P < 0.05). Moreover, unsexed semen from this bull yielded lower blastocyst (P < 0.05) and blastocyst hatching rates than unsexed sperm from the other bull. CMA3-positive staining was negatively correlated with cleavage (R2 85.1, P = 0.003) and blastocyst hatching (R2 87.6, P = 0.006) rates in unsexed semen. In conclusion, while the sex-sorting process had no effect on sperm protamine content, we observed a bull effect for sperm protamination, which correlated to embryo development rates following IVF.

Contributions of RNA-seq to improve in vitro embryo production (IVP).

In Vitro Embryo Production (IVP) is widely used to improve the reproductive efficiency of livestock animals, however increasing the embryo development rates and pregnancy outcomes is still a challenge for some species. Thus, the lack of biological knowledge hinders developing specie-specific IVP protocols. Therefore, the contributions of RNA-seq to generate relevant biological knowledge and improve the efficiency of IVP in livestock animals are reviewed herein.

Effect of cortisol on bovine oocyte maturation and embryo development in vitro.

Glucocorticoids (GCs) are important mediators of key cellular events. Herein, we investigated the effect of adding cortisol to the IVM medium on the acquisition of developmental competency in bovine oocytes. Cortisol (0.01, 0.1, or 1 µg/mL) had no effect on cleavage rates or cell numbers of resulting blastocysts; however, supplementation with 0.1 µg/mL during IVM increased blastocyst rates of in vitro-fertilized bovine oocytes as compared to untreated controls (41 ± 10% vs. 21 ± 1.2%, P < 0.05, respectively). This concentration was chosen to assess changes in the relative expression of potential GC target genes. Oocytes matured in the presence of cortisol and their corresponding cumulus cells did not show changes in expression for genes analyzed as compared to untreated controls. Notably, blastocysts from oocytes matured in cortisol-supplemented medium expressed higher relative levels of glucose transporter 1 (GLUT1), fatty acid synthase (FASN), and heat shock protein 70 (HSP70). This study supports a role for cortisol in the acquisition of bovine oocyte competence. This is evidenced by increased blastocyst development rates and presumably related to elevated embryonic transcripts with roles in glucose and lipid metabolism, as well as the cellular response to stress.

Supplementation of bovine embryo culture medium with L-arginine improves embryo quality via nitric oxide production.

Nitric oxide (NO) is a cell-signaling molecule that regulates a variety of molecular pathways. We investigated the role of NO during preimplantation embryonic development by blocking its production with an inhibitor or supplementing in vitro bovine embryo cultures with its natural precursor, L-arginine, over different periods. Endpoints evaluated included blastocyst rates, development kinetics, and embryo quality. Supplementation with the NO synthase inhibitor N-Nitro-L-arginine-methyl ester (L-NAME) from Days 1 to 8 of culture decreased blastocyst (P < 0.05) and hatching (P < 0.05) rates. When added from Days 1 to 8, 50 mM L-arginine decreased blastocyst rates (P < 0.001); in contrast, when added from Days 5 to 8, 1 mM L-arginine improved embryo hatching rates (P < 0.05) and quality (P < 0.05) as well as increased POU5F1 gene expression (P < 0.05) as compared to the untreated control. Moreover, NO levels in the medium during this culture period positively correlated with the increased embryo hatching rates and quality (P < 0.05). These data suggest exerts its positive effects during the transition from morula to blastocyst stage, and that supplementing the embryo culture medium with L-arginine favors preimplantation development of bovine embryos.

Influence of L-arginine during bovine in vitro fertilization.

The objective of this work was to evaluate the effect of using L-arginine during in vitro fertilization (IVF) on in vitro embryonic development using Bos taurus and Bos indicus semen. Effect of different concentrations (0, 1, 10 and 50 mM) of L-arginine, added to the IVF medium, was evaluated on the fertilization rate at 18 h post-fertilization (hpf), NO3(-)/NO2(-) production during IVF by the Griess colorimetric method (30 hpf), cleavage and blastocyst rates (on Day 2 and Day 7 of culture, respectively) and total blastocyst cell number (Day 7 of culture). The results reveal that the addition of 50 mM L-arginine to IVF medium, with either Bos taurus or Bos indicus spermatozoa, decreased the cleavage rate and blastocyst rate compared to the control group. Other concentrations did not affect embryo production. However, 1 mM L-arginine with Bos indicus semen increased the proportion of hatched blastocysts. These results indicate that high L-arginine concentrations may exhibit toxic effects on bovine gametes during in vitro fertilization.