Compensability of Enhanced Cytoplasmic Droplet Rates in Boar Semen: Insights of a Retrospective Field Study.

Simple SummaryThe loss of sperm quality during liquid preservation of boar semen may depend on the initial characteristics of the raw semen. Retained cytoplasmic droplets (CD) at the sperm tail caused by incomplete sperm maturation in the epididymis are the most common morphological abnormality in boar semen. The aim of the present study was to examine whether the presence of CD affects the quality of boar sperm during long-term liquid storage. For this, sperm integrity and function were evaluated in semen samples containing different amounts of sperm with CD. It was found that the ability of sperm to perform an essential preparative step for fertilization, named capacitation, was impaired in semen samples rich in CD. Moreover, the enhanced presence of CD caused a higher incidence of sperm destabilization in semen doses preserved for long term (96 h). In contrast, sperm motility and viability were not affected by the presence of CD. The proportion of sperm with CD did not decrease during semen storage in the semen extender. It is concluded that a disturbed maturation of sperm in the epididymis impairs essential steps of fertilization and causes a faster sperm destabilization during in vitro storage.Retained cytoplasmic droplets (CD) are the most frequent sperm abnormality in boar semen. A high incidence of CD is associated with subfertility, but the underlaying reasons are not well understood. The storage of extended semen might augment the adverse effects of CD on essential steps towards fertilization, such as capacitation. The aim of this study was to examine whether the enhanced presence of CD in boar semen influences sperm’s response to the capacitation stimulus bicarbonate during long-term semen storage. Extended semen samples (n = 78) from 13 artificial insemination centers were analyzed using a flow cytometric calcium influx assay. Samples with >15% of CD showed a reduced specific response to bicarbonate and a higher non-specific destabilization after storage for 96 h and subsequent incubation at 38 °C in three variants of Tyrode’s medium (p < 0.05). The size of the bicarbonate-responsive sperm population was inversely correlated with the presence of CD-bearing sperm (r = −0.61, p < 0.01). Samples with ≤15% and samples with >15% of CD did not differ in motility or viability and acrosome integrity during semen storage. In conclusion, incomplete epididymal sperm maturation impairs the in vitro capacitation ability and promotes sperm destabilization in stored boar semen.

Isolation and characterization of boar spermatozoa with and without a cytoplasmic droplet

This single cell study aimed to clarify whether an elevated incidence of sperm with a retained cytoplasmic droplet (CD) can be compensated by a higher sperm number in boar semen doses to maintain fertility. Cluster analysis of motile spermatozoa (ten boars) revealed that spermatozoa with a CD are underrepresented in the fast, linearly moving sperm cohort compared to morphologically normal sperm. Nonetheless, the response to the motility stimulator procaine was barely affected in spermatozoa with distal CD (Cramer’s V = 0.14), but moderately affected in sperm with proximal CD (V = 0.28). Viability was lower in sperm with distal CD (p < 0.05) but not with proximal CD compared to normal sperm during 168 h storage of extended semen samples (n = 11) and subsequent thermic stress. Morphologically normal sperm from normospermic samples (n = 10) or samples with a high incidence (≥ 15%) of sperm with CD (n = 9) had similar motility patterns and responses to procaine. The origin of morphologically normal sperm had no effect on sperm viability (p > 0.05; n = 26). In conclusion, a moderately enhanced prevalence of sperm with CD seems to be compensable by an increase in sperm numbers in boar semen doses.

Once deposited in the female tract, sperm face a series of challenges that must be overcome to ensure the presence of an adequate normal sperm population close to the site of fertilization. Our aim was to evaluate the influence of the uterine milieu on boar sperm morphology. In experiment 1, sperm morphology was evaluated in the backflow (60 min after insemination) and within the uterotubal junction (UTJ) (collected ~24 h after insemination) following intrauterine sperm deposition (n = 6) and compared with the morphology of the sperm in the insemination dose. In experiment 2, the influence of the uterine fluid (UF) on sperm morphological modifications was evaluated. For this purpose, ejaculated (n = 4) and epididymal (n = 4) sperm were in vitro incubated with or without UF for 2 and 24 h. In both experiments, sperm were classified as normal, having a cytoplasmic droplet (proximal or distal) or having tail defects. The results of experiment 1 pointed to anincrease in morphologically abnormal sperm collected in the backflow (27.70%) and a reduction of the same in the UTJ (2.12%) compared with the insemination dose (17.75%) (P < 0.05). In experiment 2, incubation of ejaculated sperm with UF did not provoke any morphological modifications; however, when epididymal sperm were incubated with UF, a pronounced increase in the percentage of normal sperm was evident after 24 h compared with the initial dose (from 25.77% to 53.58%, P < 0.05), mainly due to distal cytoplasmatic droplet shedding (53.22 vs. 20.20%). In conclusion, almost all the sperm that colonize the UTJ had a normal morphology, with part of the abnormal sperm having been discarded in the backflow and part selected/modified on their way to the oviduct. UF seems to influence cytoplasmic distal droplet removal, as demonstrated previously in seminal plasma.

Economic weights for sow productivity traits in nucleus pig populations

The influence of the number and quality of spermatozoa inseminated on litter size in swine is examined in this paper. There is evidence to support the following observations. Litter size varies among boars when insemination doses contain the same numbers of spermatozoa. Increasing the number of sperm inseminated generally has a positive effect on the number of pigs born alive, especially between the range of 1 to 3 × 109 cells. The manner in which litter size responds to increasing the number of spermatozoa inseminated varies among boars. These relationships between the number of sperm inseminated and the resulting litter size provide credence to the idea that boars exhibit unique fertility patterns. These divergent fertility patterns probably reflect variability in the ability of spermatozoa from different boars to fertilize ova. A number of semen quality tests have been developed to estimate the fertility of semen. Several of these have documented that increases in estimates in sperm quality are associated with increases in litter size. However, the relative effectiveness of each of these for determining the optimal number of spermatozoa that should be included in insemination doses remains to be elucidated. In summary, increasing the fertilization rate of boars should be possible by improving semen quality, increasing the number of spermatozoa inseminated, and adjusting using estimates of sperm quality to adjust number of sperm inseminated. However, the magnitude of changes in litter size resulting from these strategies is likely to vary considerably among boars.

There is a discrepancy between the use of terminology employed by clinicians and basic scientists concerning the cytoplasmic droplets of sperm. Most clinicians consider their presence on sperm to be indicative of abnormal sperm, whereas basic scientists consider them to be attributes of normal sperm. The presence of cytoplasmic droplets on human sperm was examined using conventional air-dried, fixed and stained sperm smears and in living and fixed wet preparations. Cytoplasmic droplets were found on the majority of motile sperm and in fixed preparations but only half of them were found in air-dried smears. There was no relationship between the presence of abnormally large cytoplasmic droplets, indicative of abnormal sperm, and the droplets found on living cells. The term 'cytoplasmic droplet' is confusingly used to describe two different sperm structures: large amounts of retained, excessive cytoplasmic remnants, that survive the air-drying procedure and are observed on abnormal sperm in conventionally stained sperm smears, and osmotically sensitive vesicles that are present on normal living sperm. A plea is made to retain the term 'cytoplasmic droplet' for the latter structure of normal sperm and to use the term 'excess residual cytoplasm' to describe the abnormally retained cytoplasm observed on abnormal sperm in smears.

Litter or clutch size is an important component of an organism's total reproductive effort. Because of its importance, the evolution of litter or clutch size has received considerable theoretical attention (e.g., Brockelman, 1975; Cody, 1966; Lack, 1948; Mountford, 1968; Ricklefs, 1970; and Smith and Fretwell, 1974). Most authors point out that optimal clutch sizes are the products of diverse selective pressures, including levels of resource abundance, the intensity of intraand interspecific competition, predation pressure, and other sources of mortality. In the face of these diverse pressures, organisms can produce clutches containing a relatively large number of offspring each of small size or clutches containing a smaller number of relatively large offspring. The balance that is struck between offspring size and number often depends on the relative importance of the selective pressures determining both parental and offspring fitness. In highly competitive environments, for example, selection will favor increased parental investment per offspring and decreased litter or clutch size whereas in environments in which predation and/or climatic changes cause heavy mortality, reduced parental investment per offspring and increased litter or clutch size will be selectively favored (Brockelman, 1975). Behavioral complexity can also influence the evolution of clutch or litter size. In vertebrates, large clutches of small hatchlings often occur in fish, amphibians, and reptiles that lack parental care whereas small clutches or litters of larger young occur in birds and mammals in which parental care is usually much more highly developed. In this paper we examine experimentally some of the factors that may determine optimal litter size in the common North American forest-dwelling cricetid rodent Peromyscus leucopus. Results of a number of studies (summarized in Fleming (1970) and Hill (1972)) indicate that average litter size in this species, whose adults weigh 22-25 g, ranges from 4.1 to 5.5. Part of this variation is geographic (Smith and McGinnis, 1968) and part is age (parity)-related (Drickamer and Vestal, 1973). In laboratory colonies of P. leucopus noveboracensis, the subspecies with which we worked, litter size averages 4.7 (Drickamer and Vestal, 1973; Hill, 1972). The basic question we set out to answer was: Is a litter size of 4.7 (or its closest integer value, 5) most fit? Does it produce the greatest number of surviving and reproducing offspring, as Lack (1948), but not Mountford (1968), would predict? In attempting to answer this question, we have examined the effect of litter size and female parity on the following parameters: (1) birth weight, (2) survivorship and growth rates of young during their first four weeks of life, (3) weight at weaning, and (4) the ability of weanlings to survive exposure to acute stress.

Fertilizing capacity of boar semen diluted with Beltsville TS (BTS) and Modified Kiev (MK) extenders in relation to storage time and number of spermatozoa per insemination dose

Background: Moringa leaf extract (MLE) is one of the natural ingredients that can improve the quality of preserved semen by preventing sperm lipid oxidation and increase the sperm storage time. The aim of the present study is to evaluate the effect of Moringa oleifera leaves extract on boar semen quality during short-term preservation at liquid state. Methods: Four mature healthy Large White Yorkshire boars of about 1.5-2 years age group having good reproductive health was selected for the study. Ten ejaculates were collected from each boar. Each ejaculate was divided into 4 aliquots (equal volume and sperm concentration) and Moringa oleifera leaves extract (MLE) @ 0 µg/ml (control), 100 µg/ml, 200 µg/ml and 300 µg/ml w/v was mixed in each aliquot after extension of semen samples with beltsville thaw solution (BTS) extender. The semen was evaluated for initial motility, percent live sperm, sperm abnormalities, sperm membrane integrity, acrosomal integrity and level of lipid peroxidation. Ten gilts/sows were artificially inseminated in each group with the liquid preserved semen to record the conception rates and litter size. Result: The percent sperm motility, live sperm, plasma membrane integrity and acrosomal integrity of boar spermatozoa were decreased significantly (P less than 0.01) while preserving the semen sample by using BTS extenders along with 100 µg/ml,200 µg/ml and 300 µg/ml Moringa oleifera leaves extract at 18°C for 48 hours. The mean percent of motile sperm, live sperm, intact plasma membrane, intact acrosome among the groups i.e., 100 µg/ml, 200 µg/ml and 300 µg/ml Moringa oleifera leaves extract were increased significantly (P less than 0.01) as compared to control at 24 and 48 hours of preservation. The mean percentage of sperm abnormalities and lipid peroxidation among the groups i.e., 100 µg/ml, 200 µg/ml and 300 µg/ml Moringa oleifera leaves extract were increased significantly (P less than 0.05) at 0, 24 and 48 (P less than 0.01) at hours of preservation as compared to control group. The supplementation of Moringa oleifera leaves extract in BTS extender at concentration of 100 µg/ml, 200 µg/ml and 300 µg/ml significantly improved the Large White Yorkshire boar’s semen quality during short term preservation at 18°C as compared to control. The conception rate and average litter size at birth among the groups were increased significantly (P less than 0.01) as compared to control (C). The significantly (P less than 0.01) higher conception rate and litter size at birth was recorded in T3 group i.e. 300 µg/ml Moringa oleifera leaves extract supplemented semen. In conclusion, the concentration of 300 µg/ml Moringa oleifera leaves extract was found to be better among other concentrations.

Small RNA expression patterns in seminal plasma exosomes isolated from semen containing spermatozoa with cytoplasmic droplets versus regular exosomes in boar semen

Histochemical and ultrastructural evaluations of cytoplasmic lipid droplets in bovine embryos cultured in serum-free and serum-containing media

Sperm cytoplasmic droplets (CDs) are remnants of cytoplasm that can cause a number of problems if it not shed from the sperm after ejaculation. Exosomes can rapidly bind to sperm, but it is not clear whether exosomes can affect the migration and shedding of CDs. We first extracted and characterized seminal plasma exosomes from boar semen containing sperm with low or high rates of CDs. Then, the transcriptomic and proteomic detection of these exosomes were performed to analyze the differences between the two groups of seminal plasma exosomes. The results revealed that 486 differentially expressed genes (DEGs), 40 differentially expressed proteins (DEPs), and 503 differentially expressed lncRNAs (DElncRNAs) were identified between the low CD rate group and high CD rate group. Integrative multi-omics analysis showed that exosome components may affect migration and shedding of cytoplasmic droplets by influencing cytoskeletal regulation and insulin signaling, including regulation of the actin cytoskeleton, ECM-receptor interaction, axon guidance, insulin secretion, and the insulin signaling pathway. Overall, our study systematically revealed the DEGs, DEPs, and DElncRNAs in seminal plasma exosomes between low CD rate semen and high CD rate semen, which will help broaden our understanding of the complex molecular mechanisms involved in the shedding of CDs.

Impact of holding and equilibration time on post-thaw quality of shipped boar semen

The effects of postnatal litter size on pre- and postweaning growth, daughter's litter size and rebreeding performance of the dam were examined in rats that had undergone 14 generations of selection for rate (LG) or efficiency (LE) of postweaning protein gain followed by relaxed selection, and in a randomly selected control line (C). Data came from generations 23 to 24 (standardized litters) and 25 to 26 (unstandardized litters). Generation 25 dams were remated at weaning for two additional parities. Regressions of average pup weight on average postnatal litter size were negative and increased in magnitude from birth to weaning, but decreased with parity number. Average litter size from birth to weaning (3 wk) affected postweaning weights at 5 and 7 wk, but compensatory growth occurred, especially during wk 3 to 5. The effect of dam's litter size (the litter in which the daughter was born) on daughter's weight was still negative and significant at mating and affected litter size of daughters. This negative maternal effect on daughter's litter size was larger when the dam's litter was not standardized. Litter size weaned also affected rebreeding interval of females.