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.

Fe 2C /ascorbate, hydrogen peroxide (H 2 O 2 ), and hypoxanthine/xanthine oxidase (XOD) are commonly used for inducing oxidative stress on spermatozoa. A comparative study of these agents was carried out on thawed spermatozoa from red deer. First, we tested a high, medium, and low concentration of each agent: 100, 10, and 1 mM Fe 2C (hydroxyl radical generator); 1 mM, 100, and 10 mM H 2 O 2 ; and 100, 10, and 1 mU/ml XOD (superoxide and H 2 O 2 generator), incubated at 37 8C for 180 min. Intracellular reactive oxygen species (ROS; H 2 DCFDA) increased with dose and time similarly for the three systems at each concentration level. Motility and mitochondrial membrane potential (Dj m ) were considerably decreased by H 2 O 2 (1 mM and 100 mM) and XOD (100 and 10 mU/ml

In the present study, we have related mitochondrial membrane potential (DeltaPsim) and forward scatter (FSC) to apoptotic-related changes in spermatozoa. Thawed red deer spermatozoa were incubated in synthetic oviductal fluid medium (37 degrees C, 5% CO2), with or without antioxidant (100 microm Trolox). At 0, 3, 6 and 9 h, aliquots were assessed for motility and were stained with a combination of Hoechst 33342, propidium ioide (PI), YO-PRO-1 and Mitotracker Deep Red for flow cytometry. The proportion of spermatozoa YO-PRO-1+ and PI+ (indicating a damaged plasmalemma; DEAD) increased, whereas that of YO-PRO-1- and PI- (INTACT) spermatozoa decreased. The proportion of YO-PRO-1+ and PI- spermatozoa (altered plasmalemma; APOPTOTIC) did not change. Both DEAD and APOPTOTIC spermatozoa had low DeltaPsim. Most high-DeltaPsim spermatozoa were INTACT, and their proportion decreased with time. The FSC signal also differed between different groups of spermatozoa, in the order APOPTOTIC > DEAD > INTACT/low DeltaPsim > INTACT/high DeltaPsim; however, the actual meaning of this difference is not clear. APOPTOTIC spermatozoa seemed motile at 0 h, but lost motility with time. Trolox only slightly improved the percentage of INTACT spermatozoa (P < 0.05). The population of APOPTOTIC spermatozoa in the present study may be dying cells, possibly with activated cell death pathways (loss of DeltaPsim). We propose that the sequence of spermatozoon death here would be: (1) loss of DeltaPsim; (2) membrane changes (YO-PRO-1+ and PI-); and (3) membrane damage (PI+). INTACT spermatozoa with low DeltaPsim or altered FSC may be compromised cells. The present study is the first that directly relates membrane integrity, apoptotic markers and mitochondrial status in spermatozoa. The results of the present study may help us understand the mechanisms leading to loss of spermatozoon viability after thawing.

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: 67.462.0 vs 57.162.8; NAR: 67.162.5 vs 54.563.5; viability: 68.862.0 vs 60.162.8; HOST: 71.362.2 vs 63.163.1). 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 mm 2 vs 34.42 mm 2 ). 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.

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ABSTRACT: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 37uC. 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 37uC 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.

ABSTRACT: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 Triladylா-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 (37ЊC for 20 seconds), II (60ЊC for 8 seconds) and III (70ЊC 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 Ͻ .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 Ͻ .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 ϭ .014) when spermatozoa were thawed at 37ЊC for 20 seconds than were warmed at 60ЊC 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 frozenthawed deer epididymal spermatozoa were dependent on warming rates, but each stag exhibited its own sensitivity to cryopreservation.

Over the past decade, there has been increasing interest in the application of reproductive technology to the conservation and management of natural populations of deer. The application of assisted reproduction technologies within natural population of deer is in its infancy. However, its future potential is enormous, particularly in relation to genetic management or conservation. This paper reviews the present state of such technologies for a wild subspecies of red deer, the Iberian red deer (Cervus elaphus hispanicus), by discussing the major components of oestrous synchronization, semen collection/cryopreservation and insemination techniques. In addition, findings made during the course of studies on natural populations have enormous potential for the understanding of novel reproductive mechanism that may not be uncovered by livestock or human studies. A summary of these results are also reviewed here.