A stallion spermatozoon's journey through the mare's genital tract: In vivo and in vitro aspects of sperm capacitation.

Conventional in vitro fertilization is not efficacious when working with equine gametes. Although stallion spermatozoa bind to the zona pellucida in vitro, these gametes fail to initiate the acrosome reaction in the vicinity of the oocyte and cannot, therefore, penetrate into the perivitelline space. Failure of sperm penetration most likely relates to the absence of optimized in vitro fertilization media containing molecules essential to support stallion sperm capacitation. In vivo, the female reproductive tract, especially the oviductal lumen, provides an environmental milieu that appropriately regulates interactions between the gametes and promotes fertilization. Identifying these 'fertilization supporting factors' would be a great contribution for development of equine in vitro fertilization media. In this review, a description of the current understanding of the interactions stallion spermatozoa undergo during passage through the female genital tract, and related specific molecular changes that occur at the sperm plasma membrane is provided. Understanding these molecular changes may hold essential clues to achieving successful in vitro fertilization with equine gametes.

Bicarbonate-Stimulated Membrane Reorganization in Stallion Spermatozoa.

Classical in vitro fertilization (IVF) is still poorly successful in horses. This lack of success is thought to be due primarily to inadequate capacitation of stallion spermatozoa under in vitro conditions. In species in which IVF is successful, bicarbonate, calcium, and albumin are considered the key components that enable a gradual reorganization of the sperm plasma membrane that allows the spermatozoa to undergo an acrosome reaction and fertilize the oocyte. The aim of this work was to comprehensively examine contributors to stallion sperm capacitation by investigating bicarbonate-induced membrane remodelling steps, and elucidating the contribution of cAMP signalling to these events. In the presence of capacitating media containing bicarbonate, a significant increase in plasma membrane fluidity was readily detected using merocyanine 540 staining in the majority of viable spermatozoa within 15 min of bicarbonate exposure. Specific inhibition of soluble adenylyl cyclase (sAC) in the presence of bicarbonate by LRE1 significantly reduced the number of viable sperm with high membrane fluidity. This suggests a vital role for sAC-mediated cAMP production in the regulation of membrane fluidity. Cryo-electron tomography of viable cells with high membrane fluidity revealed a range of membrane remodelling intermediates, including destabilized membranes and zones with close apposition of the plasma membrane and the outer acrosomal membrane. However, lipidomic analysis of equivalent viable spermatozoa with high membrane fluidity demonstrated that this phenomenon was neither accompanied by a gross change in the phospholipid composition of stallion sperm membranes nor detectable sterol efflux (p > 0.05). After an early increase in membrane fluidity, a significant and cAMP-dependent increase in viable sperm with phosphatidylserine (PS), but not phosphatidylethanolamine (PE) exposure was noted. While the events observed partly resemble findings from the in vitro capacitation of sperm from other mammalian species, the lack of cholesterol removal appears to be an equine-specific phenomenon. This research will assist in the development of a defined medium for the capacitation of stallion sperm and will facilitate progress toward a functional IVF protocol for horse gametes.

In-cell structures of conserved supramolecular protein arrays at the mitochondria-cytoskeleton interface in mammalian sperm.

Mitochondria-cytoskeleton interactions modulate cellular physiology by regulating mitochondrial transport, positioning, and immobilization. However, there is very little structural information defining mitochondria-cytoskeleton interfaces in any cell type. Here, we use cryofocused ion beam milling-enabled cryoelectron tomography to image mammalian sperm, where mitochondria wrap around the flagellar cytoskeleton. We find that mitochondria are tethered to their neighbors through intermitochondrial linkers and are anchored to the cytoskeleton through ordered arrays on the outer mitochondrial membrane. We use subtomogram averaging to resolve in-cell structures of these arrays from three mammalian species, revealing they are conserved across species despite variations in mitochondrial dimensions and cristae organization. We find that the arrays consist of boat-shaped particles anchored on a network of membrane pores whose arrangement and dimensions are consistent with voltage-dependent anion channels. Proteomics and in-cell cross-linking mass spectrometry suggest that the conserved arrays are composed of glycerol kinase-like proteins. Ordered supramolecular assemblies may serve to stabilize similar contact sites in other cell types in which mitochondria need to be immobilized in specific subcellular environments, such as in muscles and neurons.

The multi-scale architecture of mammalian sperm flagella and implications for ciliary motility.

Motile cilia are molecular machines used by a myriad of eukaryotic cells to swim through fluid environments. However, available molecular structures represent only a handful of cell types, limiting our understanding of how cilia are modified to support motility in diverse media. Here, we use cryo-focused ion beam milling-enabled cryo-electron tomography to image sperm flagella from three mammalian species. We resolve in-cell structures of centrioles, axonemal doublets, central pair apparatus, and endpiece singlets, revealing novel protofilament-bridging microtubule inner proteins throughout the flagellum. We present native structures of the flagellar base, which is crucial for shaping the flagellar beat. We show that outer dense fibers are directly coupled to microtubule doublets in the principal piece but not in the midpiece. Thus, mammalian sperm flagella are ornamented across scales, from protofilament-bracing structures reinforcing microtubules at the nano-scale to accessory structures that impose micron-scale asymmetries on the entire assembly. Our structures provide vital foundations for linking molecular structure to ciliary motility and evolution.

In vitro evaluation of cryopreserved bovine sperm and its relation to field fertility in fixed-time artificial insemination.

This study aimed to assess characteristics of bovine cryopreserved sperm and evaluate its relation to field fertility in fixed-time artificial insemination (FTAI). Semen samples of 16 bulls were used to inseminate 811 Nellore cows, and four of these bulls were also used to inseminate 101 Nellore heifers. Samples of the same ejaculate used for FTAI from each bull were analysed in the laboratory after thawing. Sperm motility and vigour were subjectively assessed by light microscope, and integrity of the plasma and acrosome membranes, and H2 O2 production were evaluated by flow cytometer. Relation among sperm characteristics and pregnancy rate of cows and heifers were evaluated by univariate and multivariate logistic regression. Subjective sperm motility and vigour did not affect the probability of pregnancy in cows or heifers. In univariate analysis for pregnancy in cows, sperm traits related to acrosome injury positively affected probability of pregnancy mainly when associated with plasma membrane integrity; H2 O2 production seems to be less important than plasma membrane integrity in affecting probability of pregnancy. In multivariate analysis, sperm traits related to injured acrosome positively affected probability of cow and heifer pregnancies while intact acrosome was negatively related to cow pregnancy. Intact plasma membrane and high H2 O2 production were positively related to cow pregnancy but negatively related to heifer pregnancy. Results suggest that a capacitation-like status of the acrosome may benefit probability of pregnancy in cows.

Relationship of testicular biometry with semen variables in breeding soundness evaluation of Nellore bulls.

This study aimed to evaluate the correlation between testicular biometry and semen variables, as well as, to relate testicular variables to the probability of selecting Nellore bulls with desirable sperm morphology when conducting breeding soundness evaluations (BSE). A total of 2055 BSEs from 506 bulls comprised the dataset. Biometric variables evaluated were: scrotal circumference, testicular volume, width, length, ratio and eccentricity; and semen variables were sperm motility, major sperm defects, minor sperm defects and normal sperm. Data of testicular biometry were correlated with data for semen variables using the Pearson's correlation assessment. Effects of testicular variables in selecting for sperm morphology of bulls in the BSE were evaluated by logistic regression. Scrotal circumference, testicular volume, length and width were positively correlated to sperm motility (0.18 to 0.19) and normal sperm (0.24 to 0.27) and negatively correlated with values for major defects (-0.24 to -0.27), but for testicular ratio and eccentricity there were coefficients near zero for all semen traits. Testicular ratio and eccentricity were not suitable for predicting the probability of selecting a bull based on semen variables using the BSE, but scrotal circumference, testicular volume, length and width were highly significant (P < 0.0001) with moderate values of area under ROC (Receiver Operating Characteristics) curve (0.608 to 0.620).

Can scrotal circumference-based selection discard bulls with good productive and reproductive potential?

Nonlinear mixed models were used to describe longitudinal scrotal circumference (SC) measurements of Nellore bulls. Models comparisons were based on Akaike's information criterion, Bayesian information criterion, error sum of squares, adjusted R2 and percentage of convergence. Sequentially, the best model was used to compare the SC growth curve in bulls divergently classified according to SC at 18-21 months of age. For this, bulls were classified into five groups: SC < 28cm; 28cm ≤ SC < 30cm, 30cm ≤ SC < 32cm, 32cm ≤ SC < 34cm and SC ≥ 34cm. Michaelis-Menten model showed the best fit according to the mentioned criteria. In this model, ß1 is the asymptotic SC value and ß2 represents the time to half-final growth and may be related to sexual precocity. Parameters of the individual estimated growth curves were used to create a new dataset to evaluate the effect of the classification, farms, and year of birth on ß1 and ß2 parameters. Bulls of the largest SC group presented a larger predicted SC along all analyzed periods; nevertheless, smaller SC group showed predicted SC similar to intermediate SC groups (28cm ≤ SC < 32cm), around 1200 days of age. In this context, bulls classified as improper for reproduction at 18-21 months old can reach a similar condition to those considered as good condition. In terms of classification at 18-21 months, asymptotic SC was similar among groups, farms and years; however, ß2 differed among groups indicating that differences in growth curves are related to sexual precocity. In summary, it seems that selection based on SC at too early ages may lead to discard bulls with suitable reproductive potential.

Parâmetros reprodutivos e produtivos em vacas leiteiras de manejo free stall / Productive and reproductive parameters of dairy cows in free stall

Avaliou-se parâmetros reprodutivos e produtivos em vacas da raça Holandês-PB no pós-parto imediato, manejadas em free stall . Utilizou-se 51 vacas entre primíparas e pluríparas. Os valores obtidos foram: escore de condição corporal ao parto 3,47±0,4; intervalos do parto à primeira ovulação 5,89±3,4 semanas; ao primeiro serviço 112,5±57,0 dias; a taxa de gestação ao primeiro serviço foi 43,6% e no período experimental 76,4%, não sendo observada diferença para ordens de parto (P>0,05). A produção de leite acumulada à concepção, produção diária leite até a concepção e produção de leite ajustado para 305 foi respectivamente, 3.335,6±1.488,4; 25,8±5,4 e 7.424,0±1.877,8 litros. Conclui-se que no manejo de Free stall , a detecção de estro, foi o principal fator limitante para alcançar melhor eficiência reprodutiva.

The reproductive and productive parameters in postpartum of 51 primiparous and pluriparous Holstein cows housed in free stall were obtained. The values measured were: body condition score at parturition (3.47±0.4), interval from parturition to first ovulation (5.89±3.4 weeks), interval parturition at first service (112.5±57.0 days), first-service pregnancy rate (43.6%) and during the experimental period (76.4%). No difference was found between parturition order (P>0.05). The accumulated milk production until conception time, daily production until conception time, and adjusted 305-day milk production was respectively 3,335.6±1,488.4; 25.8±5.4 and 7,424.0±1,877.8 liters. In free stall of this study the estrus detection was the most limiting factor to improve the reproductive efficiency.