Mayra Elena Ortiz Ávila Assumpção

Sperm quality influences bovine in vitro embryo production (IVEP). Linear regression is a statistical tool that models the relationship between a dependent variable and one or more independent variables. It can be used to predict outcomes, analyze trends, and understand the impact of variables. These models are useful for indicating which sperm variables most influence IVEP results, facilitating the selection of superior samples to enhance IVEP. Using early IVEP indicators, such as cleavage rate, can assist in scheduling recipient preparation. This work aimed to construct linear regression models to study the influence of a comprehensive set of sperm variables and cleavage rate on IVEP yields. A dataset comprising 51 semen batches from 23 Nellore bulls was compiled, including 26 sperm variables from computer-assisted sperm analysis (CASA) and flow cytometry per batch, with 184 IVEP procedures. The most robust predictive model had a coefficient of determination of 0.6358; furthermore, the BULL variable was the most influential predictor, yielding an independent coefficient of determination of 0.5218. Models that were exclusively founded on sperm analysis yielded meager coefficients of determination (<0.04). However, to predict the best batch from a bull, individual models achieve coefficients of determination ranging from 0.58 to 0.99. Contributions, impacts, and positive or negative correlations of various sperm variables with in vitro performance were influenced by the bull. We conclude that the BULL variable was the dominant predictor of in vitro performance, with cleavage rates serving as an early estimator of blastocyst rates. The predictive utility of analyzed sperm traits remains limited. Nonetheless, individualized models offer a valuable tool for selecting optimal batches for preferred bulls within IVEP laboratories, culminating in heightened blastocyst rates.

Research on reproductive advanced age has predominantly focused on women due to their biological clock. The impact of paternal senescence on fertility and offspring is still poorly understood. In this study, we characterized the effects of aging on sexual behavior and reproductive characteristics using male mice aged 4 months as a control, compared to mice aged 19 and 24 months. In the first experiment, we observed that reproductive interest and locomotor activity were reduced in older males, accompanied by reduced serum testosterone levels and testicular weight. We observed increased sperm morphological anomalies, protamine deficiency, and reduced sperm capacitation in aging mice. Aging also influenced cleavage, blastocyst and in vitro embryo development rates; embryo kinetics, and initial cell differentiation. In the second experiment, aging promoted smaller, lighter fetuses and a lower fetal weight ratio. In conclusion, an increased age impaired sexual behavior, sperm function, and embryo and fetal development.

Efficient spermatogenesis in mammals occurs when testicular temperature is approximately 2 to 8 °C below body temperature. Elevated testicular temperature can trigger oxidative stress and compromise sperm integrity during spermatogenesis, potentially resulting in damaged spermatozoa and male infertility. This study aimed to evaluate how heat stress affects the quantity of seminiferous tubules, and the abundance of germ cells within the seminiferous tubules. To this end, six Santa Inês rams were subjected to testicular insulation for 12 consecutive days, followed by two hemi-orchiectomies, the first 24 hours after insulation period to evaluate the immediate effect, and the second 30 days after the first hemi-orchiectomy to evaluate the late effect. Six Santa Inês rams composed the control group. Histological analyses were conducted to quantify the number of seminiferous tubules and the types of cells within them (spermatogonia, spermatocytes, and spermatids) in testicular fragments. Despite an increase in testicular temperature, no significant differences were observed in the number of seminiferous tubules. These findings probably reflect the resistance of Santa Ines rams to high environment temperatures. Regarding the abundance of cells, a decrease in spermatogonia (0.27% ± 0.06; 0.05% ± 0.03, p = 0.005) and an increase in spermatocytes (35.90% ± 1.58; 46.77% ± 4.33, p = 0.028) were observed immediately after the insulation period compared to 30 days after, the late effect. This result suggests an effect of the first hemi-orchiectomy on the remaining testicle, probably an attempt to maintain sperm production.

Summary: Supplementing embryonic culture medium with fetal bovine serum (FBS) renders this medium undefined. Glucose and growth factors present in FBS may affect the results of cell differentiation studies. This study tested the hypothesis that FBS supplementation during in vitro culture (IVC) alters cell differentiation in early bovine embryo development. Bovine embryos were produced in vitro and randomly distributed into three experimental groups at 90 h post insemination (90 hpi): the KSOM-FBS group, which consisted of a 5% (v/v) FBS supplementation; the KSOM33 group, with the renewal of 33% of medium volume; and the KSOM-Zero group, without FBS supplementation nor renewal of the culture medium. The results showed that the blastocyst rate (blastocyst/oocytes) at 210 hpi in the KSOM-FBS group was higher than in the KSOM-Zero group but not different from the KSOM33 group. There were no significant changes in metabolism-related aspects, such as fluorescence intensities of CellROX Green and MitoTracker Red or reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD+). Immunofluorescence analysis of CDX2 revealed that the lack of FBS or medium supplementation reduced the number of trophectoderm (TE) cells and total cells. Immunofluorescence analysis revealed a reduction of SOX17-positive cell numbers after FBS supplementation compared with the KSOM33 group. Therefore, we concluded that FBS absence reduced blastocyst rates; however, no reduction occurred when there was a 33% volume renewal of the medium at 90 hpi. We also concluded that FBS supplementation altered TE and primitive endoderm cell allocation during early bovine embryo development.

The association between advanced paternal age and impaired reproductive outcomes is still controversial. Several studies relate decrease in semen quality, impaired embryo/fetal development and offspring health to increased paternal age. However, some retrospective studies observed no alterations on both seminal status and reproductive outcomes in older men. Such inconsistency may be due to the influence of intrinsic and external factors, such as genetics, race, diet, social class, lifestyle and obvious ethical issues that may bias the assessment of reproductive status in humans. The use of the murine model enables prospective study and owes the establishment of homogeneous and controlled groups. This study aimed to evaluate the effect of paternal age on in vitro embryo development at 4.5 day post conception and on in vivo fetal development at 16 days of gestation. Murine females (2–4 months of age) were mated with young (4–6 months of age) or senile (18–24 months of age) males. We observed decreased in vitro cleavage, blastocyst, and embryo development rates; lighter and shorter fetuses in the senile compared to the young group. This study indicated that advanced paternal age negatively impacts subsequent embryo and fetal development.

Sperm routinary fitness evaluation is not sufficient to predict bull reproductive capacity as they present differences in fertility up to 40%. Among the defects which compromise spermatozoa functionality, new approaches consider the study of sperm chromatin, which is the core structure containing paternal genetic information. Sperm chromatin needs to be compacted to maintain the integrity of DNA, which occurs by binding nucleoproteins with high affinity to DNA. In the last stages of sperm maturation, chromatin is hyper-compacted by basic proteins called protamines in a process named protamination. In this review, we summarized intrinsic and extrinsic factors that are suggested to influence protamination in bull spermatozoa, considering old and new evidence from human and murine spermatozoa. Also, the current approaches to evaluate bull protamination and its relationship with fertility were described. Nevertheless, the physiological mechanisms of protamination are still poorly understood.