Higher temperatures lead to an increase of testicular metabolism that results in spermatic damage. Oxidative stress is the main factor responsible for testicular damage caused by heat stress. The aim of this study was to evaluate lasting effects of heat stress on ejaculated sperm and immediate or long-term effects of heat stress on epididymal sperm. We observed decrease in motility and mass motility of ejaculated sperm, as well as an increase in the percentages of sperm showing major and minor defects, damaged plasma and acrosome membranes, and a decrease in the percentage of sperm with high mitochondrial membrane potential in the treated group until one spermatic cycle. An increased enzymatic activity of glutathione peroxidase and an increase of stressed cells were observed in ejaculated sperm of the treated group. A decrease in the percentage of epididymal sperm with high mitochondrial membrane potential was observed in the treated group. However, when comparing immediate and long-term effects, we observed an increase in the percentage of sperm with low mitochondrial membrane potential. In conclusion, testicular heat stress induced oxidative stress that led to rescuable alterations after one spermatic cycle in ejaculated sperm and also after 30 days in epididymal sperm.

Action of reactive oxygen species, protamination failures and apoptosis are considered the most important etiologies of sperm DNA fragmentation. This study evaluated the effects of induced lipid peroxidation susceptibility on native semen profile and identified the mechanisms involved in sperm DNA fragmentation and testicular antioxidant defense on Santa Ines ram sperm samples. Semen was collected from 12 adult rams (Ovis aries) performed weekly over a 9-week period. Sperm analysis (motility, mass motility, abnormalities, membrane and acrosome status, mitochondrial potential, DNA fragmentation, lipid peroxidation and intracellular free radicals production); protamine deficiency; PRM1, TNP1 and TNP2 gene expression; and determination of glutathione peroxidase (GPx), glutathione reductase, catalase (CAT) and superoxide dismutase activity and immunodetection in seminal plasma were performed. Samples were distributed into four groups according to the sperm susceptibility to lipid peroxidation after induction with ascorbate and ferrous sulfate (low, medium, high and very high). The results were analyzed by GLM test and post hoc least significant difference. We observed an increase in native GPx activity and CAT immunodetection in groups with high susceptibility to induced lipid peroxidation. We also found an increase in total sperm defects, acrosome and membrane damages in the group with the highest susceptibility to induced lipid peroxidation. Additionally, the low mitochondrial membrane potential, susceptible to chromatin fragmentation and the PRM1 mRNA were increased in the group showing higher susceptibility to lipid peroxidation. Ram sperm susceptibility to lipid peroxidation may compromise sperm quality and interfere with the oxidative homeostasis by oxidative stress, which may be the main cause of chromatin damage in ram sperm.

Sperm DNA fragmentation is considered one of the main causes of male infertility. The most accepted causes of sperm DNA damage are deleterious actions of reactive oxygen species (ROS), defects in protamination, and apoptosis. Ram sperm are highly prone to those damages due to the high susceptibility to ROS and to oxidative stress caused by heat stress. We aimed to evaluate the effects of heat stress on the chromatin of ejaculated and epididymal sperm and the activation of apoptotic pathways in different cell types in ram testis. We observed higher percentages of ejaculated sperm with increased chromatin fragmentation in the heat stress group; a fact that was unexpectedly not observed in epididymal sperm. Heat stress group presented a higher percentage of spermatozoa with DNA fragmentation and increased number of mRNA copies of transitional protein 1. Epididymal sperm presented greater gene expression of protamine 1 on the 30th day of the spermatic cycle; however, no differences in protamine protein levels were observed in ejaculated sperm and testis. Localization of proapoptotic protein BAX or BCL2 in testis was not different. In conclusion, testicular heat stress increases ram sperm DNA fragmentation without changes in protamination and apoptotic patterns.

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In vitro embryo production (IVP) efficiency is reduced when compared to in vivo. The basic knowledge of bovine in vitro oocyte maturation (IVM) mechanisms provides support to improve in vitro embryo production yields. The present study assessed the effects of bone morphogenetic protein 15 (BMP15), fibroblast growth factor 16 (FGF16) and their combined action on cumulus cells (CC) expansion, oocyte and CC DNA fragmentation, oocyte nuclear maturation, energetic metabolism and progesterone production in bovine IVM. Cumulus‐oocyte complexes (COC) were matured in control or supplemented media containing BMP15 (100 ng/ml), FGF16 (10 ng/ml) or BMP15 combined with FGF16; and assessed at 0 and 22 hr of IVM. BMP15 alone or its association with FGF16 enhanced cumulus expansion. BMP15 decreased DNA fragmentation in both CC and oocytes, and improved oocyte nuclear maturation rate. In addition, BMP15 increased CC progesterone production, an effect not previously reported. The present study reinforces previous data pointing to a beneficial influence of BMP15 during IVM, while providing novel evidence that the underlying mechanisms involve increased progesterone production.

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Spermatogonial stem cells (SSCs) have important applications in treatments for infertility and animal transgenesis. However, the isolation of bovine SSCs is less efficient than for other species and it is lower for adult bulls. The goal of this study was to verify whether the relative gene expression of spermatogonia is affected by age after the laminin differential plating. Ten grams of parenchyma of testicles from pre-pubertal animals (5 months of age, n = 5) and bulls (3–4 years of age, n = 5) were minced and digested: colagenase (1 mg mL–1) for 30 min at 37°C and trypsin (2.5 mg mL–1) for 5 min at 37°C. Cells were plated (3 × 106 viable cells) in 60-mm culture dish covered with laminin (20 ng mL–1) and they were cultured for 15 min in high-humidity atmosphere with 5% of CO2 at 37°C. The adherent cells were recovered by enzymatic digestion with 0.1% trypsin for 1 min at 37°C. Viable cells were selected by the Trypan exclusion method, RNA was extracted from ~200.000 viable cells (Ilustra RNAspin Mini RNA, GE Healthcare, Waukesha, WI, USA) and cDNA synthesis was performed (SuperScript® VILO™, Invitrogen, Carlsbad, CA, USA). ITGA6 (integrin, α 6), SELP (Selectin P) and ICAM (Intercellular Adhesion Molecule 1) relative gene expression were determined by real-time RT-PCR (Mastercycler ep Realplex, Eppendorf International, Hamburg, Germany); GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) and ACTB (actin β) were used as housekeeping genes. The statistical analysis was performed by QPCR_MIXED (SAS®, SAS Institute Inc., Cary, NC, USA). An α level at 0.05 was always adopted. ITGA6 and ICAM1 relative expression weren't effected by age; respectively, P = 0.2367 and P = 0.3583. However, SELP was highly expressed in adult bulls (P = 0.0022). Previously, ICAM1 and SELP have also been shown to mark aging haematopoietic stem cells and were more highly expressed in spermatogonia from adult mice than younger. However, SELP is expressed in more differentiated spermatogenic cells such as human sperm. The expression levels of ITGA6 did not seem to be significantly altered by age, indicating that age affects only certain SSC properties. Financial support was provided by FAPESP (CEUA/FMVZ/USP 2509/2011).

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