Daily exposure to summer temperatures affects the motile subpopulation structure of epididymal sperm cells but not male fertility in an in vivo rabbit model.

High temperatures have negative effects on sperm quality leading to temporary or permanent sterility. The aim of the study was to assess the effect of long exposure to summer circadian heat stress cycles on sperm parameters and the motile subpopulation structure of epididymal sperm cells from rabbit bucks. Twelve White New Zealand rabbit bucks were exposed to a daily constant temperature of the thermoneutral zone (from 18 °C to 22 °C; control group) or exposed to a summer circadian heat stress cycles (30 °C, 3 h/day; heat stress group). Spermatozoa were flushed from the epididymis and assessed for sperm quality parameters at recovery. Sperm total motility and progressivity were negatively affected by high temperatures (P < 0.05), as were also specific motility parameters (curvilinear velocity, linear velocity, mean velocity, straightness coefficient, linearity coefficient, wobble coefficient, and frequency of head displacement; P < 0.05, but not the mean amplitude of lateral head displacement). Heat stress significantly increased the percentage of less-motile sperm subpopulations, although the percentage of the high-motile subpopulation was maintained, which is consistent with the fact that no effect was detected on fertility rates. However, prolificacy was reduced in females submitted to heat stress when inseminated by control bucks. In conclusion, our results suggest that environmental high temperatures are linked to changes in the proportion of motile sperm subpopulations of the epididymis, although fertility is still preserved despite the detrimental effects of heat stress. On the other hand, prolificacy seems to be affected by the negative effects of high temperatures, especially by altering female reproduction.

Absence of beneficial effects on rabbit sperm cell cryopreservation by several antioxidant agents.

The generation of reactive oxygen species associated with cryopreservation could be responsible for mammalian sperm damage and the limitable value of stored semen in artificial insemination. The aim of this study was to assess several antioxidant agents supplemented in a commercial freezing extender (Gent B®) in order to improve post-thaw rabbit sperm quality. Ejaculates of 26 New Zealand White rabbit bucks were collected, evaluated and frozen using a conventional protocol. Antioxidant agents were tested at different concentrations: bovine serum albumin (BSA; 5, 30 or 60 mg/ml), retinol (RO; 50, 100 or 200 µM) and retinyl (RI; 0.282 or 2.82 µg/ml). Per cent viability, morphological abnormalities and intact acrosomes were determined using eosin-nigrosin staining. Motility and progressivity were analyzed by computer-assisted sperm analysis (CASA). In general, all sperm quality parameters were negatively affected by the cryopreservation process, the largest effect seen was for total motility. The addition of antioxidant agents did not improve thaw sperm quality. Furthermore, for RI groups a significant decrease in sperm quality parameters was recorded. In conclusion, rabbit sperm quality is negatively affected by the cryopreservation process. To our knowledge this report is the first using these antioxidants to supplement rabbit freezing extender. BSA and RO at concentrations used in the study did not improve sperm quality parameters after thawing, whereas RI supplementation appeared to be toxic. More studies are required to find the appropriate antioxidants necessary and their most effective concentrations to improve rabbit post-thaw sperm quality.

Retinol improves in vitro oocyte nuclear maturation under heat stress in heifers.

Heat stress (HS) is especially harmful for bovine ovarian follicle development and oocyte competence. Furthermore, HS causes premature aging in oocytes due to high levels of reactive oxygen species (ROS), involved in the harmful effects over the oocyte maturation and the steroidogenic activity of follicular cells. In this study, the presumptive protective effects of antioxidant agents on heat-stressed oocytes were evaluated. Heifer oocytes were matured for 22 h under control (38°C) and HS conditions (41.5°C at 18-21 h of maturation). For each oocyte, nuclear stage and cortical granule (CG) distribution were evaluated. Steroidogenic activity of cumulus cells was also recorded. The antioxidant agents used in the study were: retinol (1.43 µg/ml), retinyl (0.28 µg/ml) and oleic acid (0.05 mg/ml). Based on a chi-squared test (P < 0.05), HS affected negatively the metaphase II (MII) progression and produced a premature CG exocytosis. Retinol improved the oocyte MII progression. However, retinyl and oleic acid, at the concentrations used in this study, could not counteract adverse effects of HS. A decrease in progesterone and increase in estradiol availability were observed when retinyl and oleic acid were supplemented to the maturation medium, respectively. In conclusion, retinol proved to be valuable in heat-stressed oocytes protecting nuclear maturation.

Bovine oocytes show a higher tolerance to heat shock in the warm compared with the cold season of the year.

Heat stress is especially harmful for bovine ovarian follicle development and oocyte competence. In this study, we assessed the effects of heat shock on oocyte maturation in oocytes collected during the cold (February-March; n = 114) or warm (May-June; n = 116) periods of the year. In both cases, cumulus-oocyte complexes were matured under control (38 °C) and heat shock conditions (41.5 °C, 18-21 h of maturation). For each oocyte, nuclear stage, cortical granule distribution and steroidogenic activity of cumulus cells were evaluated. Based on the odds ratio, heat-shocked oocytes were 26.83 times more likely to show an anomalous metaphase II morphology. When matured under heat shock conditions, oocytes obtained in both seasons were similarly affected in terms of nuclear maturation, whereas a seasonal effect was observed on cytoplasmic maturation. For oocytes collected during the cold season, the likelihood to show an anomalous maturation was 25.96 times higher when exposed to the heat treatment than when matured under control conditions. By contrast, oocytes collected during the warm season matured under control or heat shock did not show significant risk of showing an anomalous cytoplasmic maturation. Our findings indicate an increased rate of premature oocytes in response to heat shock as well as a higher tolerance to this stress of oocytes harvested in the warm season compared with those collected in the colder period.

Retinol might stabilize sperm acrosomal membrane in situations of oxidative stress because of high temperatures.

High temperatures have negative effects on sperm quality leading to temporary or permanent sterility. The study tried to confirm the harmful effects of high temperatures on epididymal sperm cells in comparison with other temperatures (scrotal, environmental, and refrigeration temperatures), the main objective was the assessment of the addition of retinol as an antioxidant agent to improve sperm quality parameters. Testes from 10 bulls were collected from a slaughterhouse. Sperm cells were flushed from the cauda epididymis and deferent duct and assessed for sperm quality parameters at recovery. Afterward, sperm cell samples were exposed to one of four different temperatures (4 °C, 22 °C, 32 °C, and 41.5 °C for 3 hours) in presence or absence of retinol in the storage extender. Percentages of viability and morphologic abnormalities were determined using eosin-nigrosin staining. Acrosome integrity and sperm plasma membrane integrity were assessed by fluorescence Pisum sativum agglutinin lectin (FITC-PSA) staining and the hypo-osmotic swelling test, respectively. Total and progressive motility were analyzed by computer-assisted sperm analysis. Sperm quality parameters were mainly affected by high temperatures (41.5 °C). The addition of all-trans-retinol to the storage extender did not show any effect on sperm quality parameters. However, the percentage of sperm cells with altered acrosome was significantly reduced when retinol was present in the extender under heat stress conditions (41.5 °C). In conclusion, retinol might stabilize sperm acrosomal membrane in situations of oxidative stress because of high temperatures.

Does heat stress provoke the loss of a continuous layer of cortical granules beneath the plasma membrane during oocyte maturation?

The objective of the present study was to evaluate the influence of heat stress on bovine oocyte maturation. Both nuclear stage and distribution of cortical granules (CG) were simultaneously evaluated in each oocyte. Oocyte overmaturation under standard conditions of culture was also evaluated. For this purpose, logistic regression procedures were used to evaluate possible effects of factors such as heat stress, overmaturation, replicate, CG distribution and metaphase II (MII) morphology on oocyte maturation. Based on the odds ratio, oocytes on heat stressed (HSO) and overmaturated (OMO) oocyte group were, respectively, 14.5 and 5.4 times more likely to show anomalous MII morphology than those matured under control conditions (CO). The likelihood for an oocyte of showing the CG distribution pattern IV (aging oocyte) was 6.3 and 9.3 times higher for HSO and OMO groups, respectively, than for the CO group. The risk of undergoing anomalous oocyte maturation, considering both nuclear stage and distribution of CG was 17.1 and 18 times greater in oocytes cultured in HSO and OMO groups, respectively, than those in the CO group. In conclusion, heat stress proved to be valuable in aging oocytes. Heat stress advanced age for nuclear and cytoplasmic processes in a similar form to that of oocyte overmaturation.