Influence of insemination time on the fertility of sex sorted frozen-thawed Y-sperm in red deer.

The aim of this study was to assess the effect of insemination timing on pregnancy rates in red deer (Cervus elaphus) when using sex-sorted sperm samples. Semen was collected by electroejaculation from 8 mature stags and processed to obtain: Conventional samples, following standard freezing procedures for commercial purposes; Control sorted samples, diluted and handled as per sorted samples but without being submitted to the sorter passage; and Y Sex Sorted (YSS) samples. Hinds were synchronized via intravaginal CIDR (Controlled Internal Drug Release) placement and given eCG (Folligon® PMSG Serum Gonadotrophin) on day 12, upon CIDR removal. They were then inseminated with one of each sperm treatment, at the following post-eCG intervals: I_1, 55:01-55:30 h; I_2, 55:31-56:00 h; I_3, 56:01-56:30 h; or, I_4, 56:31-57:00 h. Pregnancy rates were assessed at parturition. Average pregnancy rates were highest (P < 0.05) for Conventional samples (77.6%), but similar between YSS (49.8%) and Control sorted (51.3%) samples. However, when insemination interval was taken into account, pregnancy rates within the YSS group, pregnancy rates were 80 and 83.1% for I_1 and I_2, respectively were obtained. Notably, these rates were similar (P > 0.05) to the average pregnancy rates obtained with Conventional samples (77.6%). As expected, YSS sperm yielded 94% male offspring contrasting with the 57% males obtained with Conventional and Control sorted samples. Our findings support the importance of developing specific insemination timing protocols to improve pregnancy rates when using frozen-thawed sex-sorted sperm. These findings provide the foundation for further investigations in order to determine why the YSS sperm are able to fertilize the oocyte in a shorter period of time than the conventional samples.

Free-radical production after post-thaw incubation of ram spermatozoa is related to decreased in vivo fertility.

The aim of the present study was to evaluate the effect of sperm reactive oxygen species (ROS) production and DNA changes on male fertility. For that purpose, six rams with significantly different pregnancy rates were used; these were classified as having high fertility, i.e. 59.4% average pregnancy rate, or low fertility, i.e. 23.1% average pregnancy rate. Sperm quality was assessed after a two-step process of sample thawing followed by an incubation of 2h, either in the freezing extender (37°C) or after dilution in synthetic oviductal fluid (SOF; 38°C, 5%CO2). Sperm viability (YO-PRO-1), ROS production (5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein acetyl ester (CM-H2DCFDA)) and undamaged chromatin (sperm chromatin structure assay, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling, chromomycin A3) were evaluated by flow cytometry. Although no significant differences in sperm viability were observed, our results showed increased ROS production during incubation in the freezing extender as well as in SOF medium. Comparison between fertility groups showed significant differences in ROS production after 2h of incubation for the two treatments. Regarding DNA integrity, our results showed no significant differences either between treatments and incubation times or fertility groups. Linear regression analysis showed that ROS production determined by CM-H2DCFDA was a good indicator parameter for in vivo male fertility of SOF-incubated samples, yielding a fair correlation between both parameters (r=-0.92). These results indicate that detection of ROS production by CM-H2DCFDA and flow cytometry after 2h of incubation in SOF could be a useful procedure for predicting fertility of ram spermatozoa.

Improving the effect of incubation and oxidative stress on thawed spermatozoa from red deer by using different antioxidant treatments.

Antioxidants could improve sperm media, extending the viability of spermatozoa and protecting their DNA. The protective ability of lipoic acid, melatonin, Trolox and crocin was tested on red deer spermatozoa incubated at 37 degrees C. Cryopreserved spermatozoa were thawed and incubated with 1 mM or 0.1 mM of each antioxidant, with or without oxidative stress (100 muM Fe(2+)). Motility (CASA), viability, mitochondrial membrane potential and acrosomal status were assessed. Lipoperoxidation (malondialdehyde production), intracellular reactive oxygen species (ROS) and DNA status (TUNEL) were checked at 4 h. Incubation alone increased ROS and decreased motility. Oxidative stress intensified these effects, increasing lipoperoxidation and DNA damage. Lipoic acid had little protective effect, whereas 1 mM melatonin showed limited protection. Trolox lowered ROS and lipoperoxidation both in oxidised and non-oxidised samples. In oxidised samples, Trolox prevented DNA and acrosomal damage, and ameliorated motility. Crocin at 1 mM showed similar results to Trolox, but noticeably stimulated motility and had no effect on lipoperoxidation. In a second experiment, a broader range of crocin and melatonin concentrations were tested, confirming the effects of crocin (positive effects noticeable at 0.5-0.75 mM), but showing an increase in lipoperoxidation at 2 mM. Melatonin was increasingly effective at 2.5 and 5 mM (ROS, lipoperoxidation and DNA status). Crocin seems a promising new antioxidant, but its particular effects on sperm physiology must be further studied, especially the consequences of motility stimulation and confirming its effect on lipoperoxidation. Melatonin might be useful at relatively high concentrations, compared to Trolox.

Sperm characteristics and DNA integrity of Iberian red deer (Cervus elaphus hispanicus) epididymal spermatozoa frozen in the presence of enzymatic and nonenzymatic antioxidants.

The main goal of this study was to investigate the potential protective effects of enzymatic and nonenzymatic antioxidants on cryopreservation injuries to red deer epididymal spermatozoa. In Experiment 1, the effects on sperm freezability of the enzymatic antioxidants catalase, superoxide dismutase, and a combination thereof were studied. In Experiment 2, sperm cryoresistance was evaluated when different nonenzymatic antioxidants, such as vitamin E, vitamin C, and butylated hydroxytoluene (BHT), were added to the freezing extender. Sperm quality was judged in vitro by microscopic assessments of individual sperm motility (SMI), viability, and acrosome (ie, spermatozoa with normal apical ridges; % NAR) and membrane (by means of the HOS test) integrity. To address fully these topics, we incorporated a new set of functional sperm tests for mitochondrial function, membrane phospholipid disorder, and sperm chromatin stability. Samples were evaluated after freezing and thawing, and after a 2-hour period of incubation at 37 degrees C. The present study demonstrates that the addition of enzymatic antioxidants to freezing extenders improves sperm viability after cooling, and improves sperm motility, acrosome integrity, and mitochondrial status (P<.05) after thawing. After a 2-hour incubation period at 37 degrees C in the presence of enzymatic antioxidants, an improvement in membrane integrity (P<.05) was observed. However, when nonenzymatic antioxidants were present in the freezing diluents, no positive effects on thawed sperm parameters were noted. The chromatin stability test did not show significant differences between the treatments. We conclude that enzymatic antioxidants should be present in the early steps of cryopreservation of epididymal spermatozoa from red deer, so as to improve motility and acrosome integrity.

Male fertility and sex ratio at birth in red deer.

Efforts to test sex ratio theory have focused mostly on females. However, when males possess traits that could enhance the reproductive success of sons, males would also benefit from the manipulation of the offspring sex ratio. We tested the prediction that more-fertile red deer males produce more sons. Our findings reveal that male fertility is positively related to the proportion of male offspring. We also show that there is a positive correlation between the percentage of morphologically normal spermatozoa (a main determinant of male fertility) and the proportion of male offspring. Thus, males may contribute significantly to biases in sex ratio at birth among mammals, creating the potential for conflicts of interest between males and females.

Influence of various permeating cryoprotectants on freezability of Iberian red deer (Cervus elaphus hispanicus) epididymal spermatozoa: effects of concentration and temperature of addition.

With the aim of finding an ideal cryoprotectant (CPA) in a suitable concentration for red deer epididymal spermatozoa cryopreservation, we evaluated the effects of the 3 most commonly used CPAs, glycerol (GLY), ethylene glycol (EG), and propylene glycol (PG), on sperm cryoresistance. The aim of Experiment 1 was to evaluate the influence of 3 different final concentrations (3%, 6%, and 12%) of each CPA on sperm freezability. Sperm samples were diluted to a final sperm concentration of approximately 400 x 10(6) spermatozoa/mL with a Tris-citrate-fructose-EY extender (TCF) prior to freezing. Sperm cryosurvival was judged in vitro by microscopic assessments of individual sperm motility (SMI), viability, and plasma membrane (by means of the HOS test) and acrosome (NAR) integrities. Thawed samples were incubated at 37 degrees C for 2 hours in the freezing medium. At the end of this incubation period, sperm suspensions were again assessed. Our results showed that 12% of any CPA was toxic to red deer epididymal spermatozoa membrane integrity (P < .05). Moreover, regardless of the level of CPA, results indicated that the cryoprotective effects on red deer epididymal spermatozoa of the 3 CPAs after thawing are in the following sequence: GLY > EG > PG (higher symbols mean P < .001). Furthermore, our results also showed an improvement in sperm parameters when the TCF diluent contained 6% of GLY. In Experiment 2 extenders were prepared using GLY 6%. This experiment was designed to investigate the effect of 2 different temperatures of GLY addition -22 degrees C (ambient temperature) and 5 degrees C- on sperm freezability. Our results showed a differential response (P < .05) of motility (SMI) to temperature of GLY addition before freezing, the best being 22 degrees C (81.94 +/- 2.4% vs 72.38 +/- 2.4%). Although there were no statistically significant differences (P > .05) between the 2 temperatures of GLY addition after thawing in terms of sperm quality, after 2 hours of incubation, results tended to be better when CPAs were added at 22 degrees C. In conclusion, our work showed the efficacy of a TCF diluent with 6% of GLY and its addition at 22 degrees C, as an alternative to the more common 3%-4% of GLY and addition at 5 degrees C, in red deer semen freezing protocols.

Functional significance of the sperm head morphometric size and shape for determining freezability in iberian red deer (Cervus elaphus hispanicus) epididymal sperm samples.

In the present study, computer-automated sperm head morphometry of epididymal samples was used to determine if sperm head area and shape are useful measurements for separating "good" and "bad" Iberian red deer freezers. A microscope slide was prepared from single diluted sperm fresh samples collected from 38 mature stags. Slides were air-dried and stained with Hemacolor. The sperm head area and shape (length/width) for a minimum of 145 sperm heads were determined for each male by means of the Sperm-Class Analyser. The remainder of each sample was frozen. After thawing, sperm cryosurvival was judged in vitro by microscopic assessments of individual sperm motility and of plasma membrane and acrosome integrities. All sperm parameters evaluated at thawing were placed in a statistical database and a multivariate cluster analysis performed. Mean sperm parameters of the 2 clusters generated ("bad" and "good" freezers) were compared by ANOVA. Our results show that sperm quality at thawing for all sperm parameters evaluated was significantly higher (P < .01) for "good" freezers than for the "bad" ones (sperm motility index: 67.4 +/- 2.0 vs 57.1 +/- 2.8; NAR: 67.1 +/- 2.5 vs 54.5 +/- 3.5; viability: 68.8 +/- 2.0 vs 60.1 +/- 2.8; HOST: 71.3 +/- 2.2 vs 63.1 +/- 3.1). Additionally, differences (P < .01) in epididymal sperm head area and shape were found between "good" and "bad" freezers before freezing, with the smallest overall sperm head dimensions found in the "good" freezers group (area: 32.04 microm2 vs 34.42 microm2). Thus, the lower the sperm head area in the fresh samples, the greater the sperm cryoresistance. Our results show that the 2 groups of males also differ in sperm head shape in fresh samples (good: 1.96 vs poor: 1.72; P < .01). It is possible that sperm head area and shape influence total sperm volume, thus causing differences in heat exchange as well as in movements of water, ions, and cryoprotectants and, in turn, on sperm freezability.

The effects of different cryoprotectants and the temperature of addition on the survival of red deer epididymal spermatozoa.

With the aim of finding an ideal cryoprotectant in a suitable concentration for red deer epididymal spermatozoa conservation, we evaluated the effects of four most commonly used cryoprotectants (CPAs), Glycerol (G), Ethylene glycol (EG), Propylene glycol (PG), and Dimethyl sulfoxide (DMSO), on the sperm survival. Besides, the effects of two temperatures of CPA addition--22 degrees C (ambient temperature) and 5 degrees C--on sperm quality were also tested. For each temperature tested, sperm samples were evaluated after 0, 15, 30 and 60 min of spermatozoa exposition to CPAs. Thus, sperm quality was in vitro judged by microscopic assessments of individual sperm motility (SMI), and of plasma membrane (Viability) and acrosome (NAR) integrities. Overall, DMSO showed the highest toxicity for red deer epididymal spermatozoa, and glycerol the lowest. Thus, at 60 min of incubation SMI results showed that the toxicity to red deer epididymal spermatozoa of the four CPAs are in the following sequence: G approximately = EG approximately = PG < DMSO ('less than' symbol means P < 0.05, and approximate symbol means P = 0.08). Furthermore, our results also showed a differential response of acrosome membrane to temperature of CPAs addition. Regardless of the CPA used, statistically significant variations (P < 0.05) were found between the two temperatures of addition of CPAs for acrosome integrity, the best being 22 degrees C (NAR = 83.8% vs. 69.8%). These data indicate that sperm quality of red deer epididymal spermatozoa, in addition to be affected by the cryoprotectant, can also be influenced by the temperature at which CPAs are added prior to freezing.

Effects of thawing procedure on postthawed in vitro viability and in vivo fertility of red deer epididymal spermatozoa cryopreserved at -196 degrees C.

In this study, we have determined the effects of individual factor and thawing procedure on in vitro viability and in vivo fertility of frozen-thawed red deer epididymal spermatozoa. The spermatozoa that were collected from 13 Iberian deer stags were diluted at room temperature in a Triladyl-20% egg yolk medium and frozen in nitrogen vapors. In the first experimental series, sperm samples were collected from 10 mature stags. For thawing, the frozen straws were subjected to 3 different procedures: I (37 degrees C for 20 seconds), II (60 degrees C for 8 seconds) and III (70 degrees C for 5 seconds). Sperm cryosurvival was judged in vitro by microscopic assessments of individual sperm motility (SM) and of plasma membrane and acrosome (NAR) integrities. Statistically significant variations were found (P <.05) between stags for most of the seminal parameters evaluated. The thawing procedure did not have an effect on the seminal characteristics evaluated after this process, except for SM (P <.05), with the best overall recovery rates after freezing and thawing found with the use of protocol I. Our results also show a differential resistance to return to isosmotic conditions of spermatozoa thawed using the different thawing protocols. In the second experimental series (insemination artificial trial), with spermatozoa from 3 stags, results of fertility were statistically higher (69.7% vs 42.4%, P =.014) when spermatozoa were thawed at 37 degrees C for 20 seconds than were warmed at 60 degrees C for 8 seconds. Therefore, thawing protocol I, which provides slow thawing rates, was the most beneficial for epididymal spermatozoa thawing of the cervid subspecies analyzed in this study. In summary, high in vitro survival and in vivo fertility of frozen-thawed deer epididymal spermatozoa were dependent on warming rates, but each stag exhibited its own sensitivity to cryopreservation.

Head dimensions of cryopreserved red deer spermatozoa are affected by thawing procedure.

The objective of this study was to evaluate the effects of the thawing procedure on red deer spermatozoa distribution in morphologically distinct subpopulations after freezing and thawing. For this purpose, epididymal spermatozoa were thawed using two different thawing protocols (I = 37 degree celsius for 20 s vs. II = 70 degree celsius for 5 s). The spermatozoa, from 10 Iberian deer stags, were diluted at room temperature in a Triladyl-20 percent egg yolk medium and frozen in nitrogen vapor. Standard sperm freezability was judged by microscopic assessments of sperm motility. The thawing procedure had an effect on sperm motility percentage (P = 0.05), with the best overall recovery rates found with the use of protocol I (76.8 + or - 1.8 vs. 70.6 + or - 1.8). Moreover, the morphometric dimensions for a minimum of 200 sperm heads were analyzed from each sample by means of the Sperm-Class Analysez (SCA), and the mean measurements recorded. Deer sperm heads were significantly (P = 0.01) smaller when spermatozoa were thawed using protocol II than when using procedure I (area = 30.02 square micrometers vs. 30.32 square micrometers; width = 4.47 micrometers vs. 4.51 micrometers; length = 8.05 micrometers vs. 8.11 micrometers), but not for all stags. All sperm head measurements were placed in a statistical database and a multivariate cluster analysis performed. Mean measurements for all parameters of the major clusters for the two different thawing procedures were compared by ANOVA. The mean values for length, width, area, perimeter, shape factor and width/length in the major cluster of sperm head dimensions for thawing protocol I were significantly different from those for protocol II (P = 0.001). In addition, differences were found within all stags for whole morphometric parameters (P = 0.001), with the smallest overall sperm head dimensions found with the use of protocol II. Additionally, the rapid thawing protocol produced a dramatic loss of heterogeneity. Finally, our results showed that the greater the loss of heterogeneity, the greater the degree of sperm cryoinjury.