What is the relevance of seminal plasma from a functional and preservation perspective?

When preserving sperm in the liquid or cryopreserved state, seminal plasma (SP) components within ejaculates can alter fertilizing capacity of these gametes. Depending on the species or how semen is collected, volume and concentration of SP components varies considerably. The SP contains substances essential for maintenance of sperm viability and fertility; however, these components can be deleterious depending on quantity, or duration of time before there is removal of SP from sperm in semen processing. Substances that impair (e.g., BSP - bull; HSP-1 - stallion; Major seminal plasma protein PSPI - boar) or improve (e.g., spermadhesin PSP-I - boar) spermatozoa fertilizing capacity have been identified. Depending on individual males, species, and semen collection procedures, SP removal may be beneficial before preservation in the liquid or cryopreserved state. In some cases, SP that is removed can be added back to thawing extender with there being positive effects in thawed sperm and for sperm viability in the female reproductive tract. In this review article, there is a focus on different effects of SP in samples of cooled and cryopreserved semen from four domestic species (pigs, horses, cattle, and sheep) with there being emphasis on how SP modulates the function and morphology of sperm cells before, during, and after preservation in the refrigerated or cryopreserved state. The present review is part of the Festschrift in honor of Dr. Duane Garner who made major contributions to the area of focus in this manuscript as evidenced by the many times his research is cited in this manuscript.

Effects of oestrous synchronization with altrenogest in gilts on endometrial and embryonic characteristics.

The use of altrenogest (ALT) supplementation for oestrous synchronization improves subsequent reproductive performance of gilts and sows. However, the causes of this improvement in reproductive performance after ALT treatment are not fully/clearly understood. The objective of this study was to evaluate the effects of ALT supplementation for oestrous synchronization in gilts on the endometrial glands and embryonic development characteristics at 28 days of pregnancy. Pregnant gilts were divided into two experimental treatments: Control (did not receive ALT; n = 9 gilts) and ALT (ALT feeding at 20 mg/day for 18 days; n = 9 gilts). At 28 days of pregnancy, six gilts from each treatment were slaughtered, and reproductive tracts were immediately evaluated. There was no statistical difference (P > 0.05) between treatments regarding ovulation rate, number of embryos, number of vital embryos and number of non-vital embryos. Embryo weight, length and embryonic vesicle weight were lower in ALT treatment compared with Control (P < 0.01), and it was lower in the cervical uterine region compared with apex uterine region, respectively (P < 0.05). Higher values of gland duct area, gland duct perimeter, percentage of the glandular area and total endometrial area were observed in ALT treatment compared with Control (P < 0.05). The use of ALT during 18 days for oestrous synchronization in gilts increased the gland duct area, perimeter and total endometrial area but did not increase the embryo number and embryo size at day 28 of pregnancy.

Supplemental progesterone during early pregnancy exerts divergent responses on embryonic characteristics in sows and gilts.

Progesterone (P4) plays a key role in pregnancy establishment and maintenance; during early pregnancy, P4 stimulates the production and release of uterine secretions necessary for conceptus growth prior to implantation; therefore, exogenous P4 supplementation may improve embryo development. This study evaluated the effects of supplementation during early pregnancy with long-acting injectable progesterone or altrenogest on embryonic characteristics of sows and gilts. Thus, a total of 32 sows and 16 gilts were used. On day 6 of pregnancy sows and gilts were allocated to one of the following groups: non-supplemented; supplemented with 20 mg of altrenogest, orally, from days 6 to 12 of pregnancy; supplemented with 2.15 mg/kg of long-acting injectable progesterone on day 6 of pregnancy. Animals were killed on day 28 of pregnancy, and ovulation rate, embryo survival, embryo weight, crown-to-rump length, uterine glandular epithelium and endometrial vascularization were assessed. Treatments had no effect on pregnancy rate, embryo survival or endometrial vascular density (P > 0.05). Non-supplemented gilts presented larger and heavier embryos compared to gilts from supplemented groups (P < 0.05). Sows in the altrenogest group presented larger and heavier embryos compared to non-supplemented sows and sows supplemented with long-acting injectable progesterone. In conclusion, supplementation of sows and gilts with progestagen from day 6 of pregnancy can be used as a means to improve embryo survival without deleterious effects.

Dietary ractopamine supplementation of pregnant sows: what are the impacts on the neonate?

The use of additives such as ractopamine (Rac) in pregnant sows during early-mid pregnancy is an alternative to increase foetal and progeny growth and development. However, Rac supplementation in finishing pigs can lead to behavioural and physiological changes similar to the typical stress responses. The objective of this study was to evaluate the effects of dietary supplementation with Rac in pregnant sows from day 25 to 50 of gestation (pre-hyperplastic stage) on piglet's vitality, blood parameters, number, diameter and perimeter of muscle fibres in semitendinosus muscle and developmental characteristics of piglets at birth to weaning. Forty-one hybrid sows were divided into three dietary treatments: (1) control diet without Rac (control), (2) addition of 10 mg/kg of Rac (Rac10) and (3) addition of 20 mg/kg of Rac (Rac20). Higher numbers of low-vitality piglets (P<0.05) were observed in Rac-fed sows, regardless of dose, compared with the control group. Very low-density lipoprotein levels were lower in the Rac10 group when compared with the Rac20 group at day 21. Haematocrit was greater, and the mean corpuscular haemoglobin concentration was lower in piglets from Rac-fed sows. No significant statistical differences were detected regarding piglets body weight, average daily gain, blood gasometry, complete blood count and muscle fibre measurements in semitendinosus muscle. The use of Rac in pregnant sows reduced the vitality parameters of piglets but did not improve the performance from birth until weaning and did not negatively influence the haematological parameter and lipid metabolism.

Ivermectin does not interfere with seminal and hormonal parameters in male rabbits.

Ivermectin (IVM) is a macrocyclic lactone used as a broad spectrum antiparasitic agent against nematodes and arthropods. It is mainly used in the control of parasitic infections of domestic animals, and recently has been used in humans to treat onchocerciasis, scabies, and pediculosis. In mammals, evidence has indicated that macrocyclic lactones interact with gamma-aminobutyric acid (GABA)-mediated chloride channels. The GABAergic system is known to be involved in the manifestation of sexual behavior, and previous studies have shown that IVM impaired sexual behavior in both male and female rats. Thus, considering that IVM may interfere with the sexual sphere, this study evaluated the temporal (1 up 60 days) effects of exposure to IVM (0.2 and 1.0 mg/kg, administered subcutaneously) on seminal and hormonal parameters of male rabbits. In male rabbits, the spermatozoa concentration, motility and morphology, the integrity of the plasmatic, acrosomal and mitochondrial membranes of the spermatozoa, the organ weights, gonadosomatic index, serum testosterone concentrations, histopathological findings were evaluated and hematological and serum biochemical analysis was conducted. No changes were observed in male seminal parameters evaluated by spermatozoa concentration, motility, and morphology, nor the potential for fertilization evaluated by the integrity of the plasmatic, acrosomal, and mitochondrial membranes of the spermatozoa; there was also no interference in serum testosterone concentration, serum biochemistry and hematological parameters. The findings of this study using the artificial vagina for collection of semen and computer-assisted semen analysis showed that IVM at doses of 0.2 and 1.0 mg/kg of SC did not alter any of the semen parameters of rabbits evaluated for up to 60 days after administration.

Absence of seminal plasma from sperm-rich fraction decreases boar sperm quality characteristics during the course of liquid storage.

Seminal plasma (SP), the fluid that surrounds the sperm cells, is known to exert substantial influence on sperm physiology. The SP has a pivotal role in sperm function in vivo, and due to its components, it functions in an ambiguous manner in vitro, simultaneously possessing deleterious and beneficial effects. This experiment aimed to describe the differences between the presence or absence of SP from the sperm-rich fraction on some spermatozoa characteristics (kinetics, plasma and acrosome membrane integrity, lipid peroxidation and capacitation-like changes). Furthermore, this experiment focused on distinguishing the effects of SP on the variables evaluated from the effects of centrifugation during SP removal. Total and progressive sperm motility, as well as integrity of plasma and acrosome membranes, were less (P < 0.05) in the absence of SP. Membrane lipid peroxidation (P < 0.05) and sperm membrane stability (P < 0.05) did not differ among treatments. The SP from the sperm-rich fraction is important for the maintenance of adequate structural and functional characteristics of extended liquid boar semen and should be present in seminal doses throughout storage. Furthermore, the detrimental effect on the variables evaluated was caused solely by the absence of SP and not by the process of removal through centrifugation at 500 x g for 10 min.

Seminal plasma arising from the whole boar sperm-rich fraction increases the stability of sperm membrane after thawing.

Boar spermatozoa arising from the sperm-rich ejaculate fraction are reported to have a more stable plasma membrane and are more resistant to cold shock and premature acrosome reaction than spermatozoa from the whole ejaculate. Furthermore, seminal plasma (SP) can increase the cryotolerance of boar spermatozoa, and in other domestic species, it has the ability to reverse cryopreservation damage. This study aimed to evaluate the effects of boar SP arising from the whole sperm-rich ejaculate fraction (SP-SRF) on the integrity, stability, and peroxidation of sperm membranes after thawing. Each ejaculate ( = 24) was divided among 4 treatments: control (CT), centrifuged and suspended in autologous SP-SRF (CS), centrifuged with withdrawn SP-SRF (CW), and post-thawed SP arising from the whole sperm-rich fraction addition to CW (CWSP). After thawing, all treatments were incubated for 5, 60, and 120 min and were analyzed for membrane integrity, fluidity, and peroxidation by flow cytometer. The absence of SP-SRF increased the lipid disorder ( < 0.05) but had no effect on lipid peroxidation ( > 0.05) or membrane integrity ( > 0.05). However, the increase in lipid disorder by withdrawal of SP-SRF was reversed by SP-SRF addition ( < 0.05) to the post-thawing medium, whereas plasma and acrosomal membrane integrity ( > 0.05) and lipid peroxidation ( > 0.05) were unchanged. In conclusion, despite the centrifugation effects, the addition of SP arising from the whole sperm-rich fraction to post-thawed boar semen decreased sperm lipid disorder without an influence of the sperm membrane integrity and peroxidation.

Organic selenium supplementation increases PHGPx but does not improve viability in chilled boar semen.

This study evaluated the effects of dietary organic selenium (Se) on viability of chilled boar semen. Twelve boars were divided into three groups: control (CON), 0.3 mg kg(-1) sodium selenite; inorganic (INO), 0.5 mg kg(-1) sodium selenite and organic (ORG), 0.5 mg kg(-1) Se yeast. The experiment was conducted within 10 weeks, and analysis was performed fortnightly, in storage semen by 72 h. No effect was observed on motility; however, straightness and linearity percentages were higher (P < 0.05) in the animals receiving CON diet compared with INO group. Percentages of cells with both plasma and acrosomal intact membranes, lipidic membrane peroxidation and mitochondrial membrane potential were similar on all treatments. Animals receiving CON diet presented higher (P < 0.05) values of ATP when compared with INO group. The PHGPx was higher (P < 0.05) in animals that received ORG in comparison with INO group. In conclusion, organic selenium supplementation increases PHGPx but does not improve chilled semen viability in 72 h.

Gonadotropin-induced puberty does not impair reproductive performance of gilts over three parities.

The aim of this study was to evaluate the reproductive performance of three parities of gilts treated or not treated with gonadotropin to induce puberty. Sixty gilts received 600 IU of equine chorionic gonadotropin (eCG) followed by 2.5 mg of porcine luteinizing hormone (LH) 72 h later. Fifty-nine other gilts were exposed only to a mature boar for 15 min twice daily. Artificial insemination (AI) was performed at 0, 12 and 24 h after the detection of oestrus, and gestation was confirmed by ultrasound after 35 days. Sows were inseminated at the first post-weaning oestrus. The total numbers of piglets born, piglets born alive, stillborn, mummified foetuses, as well as pregnancy and farrowing rates were evaluated for each of the three parities. Culling rates, farrowing intervals and weaning-to-oestrous intervals (WEI) were also analysed. Mean age at puberty and oestrous manifestation were not significantly different between treatments (p = 0.0639; 179.20 ± 17.52 compared with 173.96 ± 16.94, 91.66% compared with 94.92%) across the experimental period. However, females that underwent puberty induction showed modest increases both in the number of total pigs born and in the number of piglets born alive. In conclusion, puberty induction through exogenous gonadotropin administration in field conditions did not induce a more concentrated first oestrous manifestation, but trended to a modest increase in the number of pigs born alive in the first parity and a reduced culling rate during the first gestation.

Effect of sequence of insemination after simultaneous thawing of multiple semen straws on conception rate to timed AI in suckled multiparous Nelore cows.

The objective was to determine the effect of sequence of insemination after simultaneous thawing of multiple 0.5 mL semen straws on conception rate in suckled multiparous Nelore cows. The effect of this thawing procedure on in vitro sperm characteristics was also evaluated. All cows (N = 944) received the same timed AI protocol. Ten straws (0.5 mL) of frozen semen from the same batch were simultaneously thawed at 36 °C, for a minimum of 30 sec. One straw per cow was used for timed AI. Frozen semen from three Angus bulls was used. Timed AI records included sequence of insemination (first to tenth) and time of semen removal from thawing bath. For laboratory analyses, the same semen batches used in the field experiment were evaluated. Ten frozen straws from the same batch were thawed simultaneously in a thawing unit identical to that used in the field experiment. The following sperm characteristics were analyzed: sperm motility parameters, sperm thermal resistance, plasma and acrosomal membrane integrity, lipid peroxidation, chromatin structure, and sperm morphometry. Based on logistic regression, there were no significant effects of breeding group, body condition score, AI technician, and sire on conception rate, but there was an interaction between sire and straw group (P = 0.002). Semen from only one bull had decreased (P < 0.05) field fertility for the group of straws associated with the longest interval from thawing to AI. However, the results of the laboratory experiment were unable to explain the findings of the field experiment. Sperm width:length ratio of morphometric analysis was the single sperm characteristic with a significant interaction between sire and straw group (P = 0.02). It was concluded that sequence of insemination after simultaneous thawing of 10 semen straws can differently affect conception rates at timed AI, depending on the sire used. Nevertheless, the effects of this thawing environment on in vitro sperm characteristics, remain to be further investigated.

Effects of bovine sperm cryopreservation using different freezing techniques and cryoprotective agents on plasma, acrosomal and mitochondrial membranes.

The success of semen cryopreservation is influenced by several factors, such as freezing curves and cryoprotectants. These two factors are of special interest once they may lead to many important physical-chemical changes resulting in different degrees of damage in spermatozoa structure. This experiment was designed to compare the effect of bull semen cryopreservation using two freezing techniques: conventional (CT--cooling rate of -0.55 °C min(-1) and freezing rate of -19.1 °C min(-1) and automated (AT--cooling rate of -0.23 °C min(-1) and freezing rate of -15 °C min(-1)), performed with different curves, and with three cryoprotectants (glycerol, ethylene glycol and dimethyl formamide) on bovine sperm motility and integrity of plasma, acrosomal and mitochondrial membranes. These variables were simultaneously evaluated using the fluorescence probes propidium iodide, fluorescein-conjugated Pisum sativum agglutinin and MitoTracker Green FM. The effects of freezing techniques, as well as of different cryoprotectants were analysed by the analysis of variance. The means were compared by Fisher's test. There were no significant differences between freezing techniques (P > 0.05). Glycerol showed higher percentages of motility, vigour and integrity of plasma, acrosomal and mitochondrial membranes than other two cryoprotectants (P < 0.05). Ethylene glycol preserved higher motility and integrity of plasma and mitochondrial membranes than dimethyl formamide (P < 0.05). Sperm motility with glycerol was 30.67 ± 1.41% and 30.50 ± 1.06%, with ethylene glycol was 21.17 ± 1.66% and 21.67 ± 1.13% and with dimethyl formamide was 8.33 ± 0.65% and 9.17 ± 0.72% to CT and AT curves, respectively. The percentage of spermatozoa with simultaneously intact plasma membrane, intact acrosome and mitochondrial function (IPIAH) was 14.82 ± 1.49% (CT) and 15.83 ± 1.26% (AT) to glycerol, 9.20 ± 1.31% (CT) and 9.92 ± 1.29% (AT) to ethylene glycol 4.65 ± 0.93% (CT) and 5.17 ± 0.87% (AT) to dimethyl formamide. Glycerol provided the best results, although nearly 85% of spermatozoa showed some degree of injury in their membranes, suggesting that further studies are required to improve the results of cryopreservation of bovine semen.

Effects of discontinuous Percoll gradient centrifugation on the quality of bovine spermatozoa evaluated with computer-assisted semen analysis and fluorescent probes association.

The objective of this study was to evaluate the quality of bovine frozen-thawed sperm cells after Percoll gradient centrifugation. Frozen semen doses were obtained from six bulls of different breeds, including three taurine and three Zebu animals. Four ejaculates per bull were evaluated before and after discontinuous Percoll gradient centrifugation. Sperm motility was assessed by computer-assisted semen analysis and the integrity of the plasma and acrosomal membranes, as well as mitochondrial function, were evaluated using a combination of fluorescent probes propidium iodide, fluorescein isothiocyanate-conjugated Pisum sativum agglutinin and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide. The procedure of Percoll gradient centrifugation increased the percentage of total and progressive sperm motility, beat frequency, rectilinear motility, linearity and rapidly moving cells. In addition, the percentage of cells with intact plasma membrane and mitochondrial membrane potential was increased in post-centrifugation samples. However, the percentage of sperm cells with intact acrosomal membrane was markedly reduced. The method used selected the motile cells with intact plasma membrane and higher mitochondrial functionality in frozen-thawed bull semen, but processing, centrifugation and/or the Percoll medium caused damage to the acrosomal membrane.

Addition of seminal plasma to post-thawing equine semen: what is the effect on sperm cell viability?

Effect of seminal plasma addition after thawing on viability or cryocapacitation is not definitively established. This experiment was performed to verify the effect of adding seminal plasma, autologous or homologous (from an animal with good semen freezability). Five ejaculates from each of four stallions with proven fertility were collected and cryopreserved. The semen was subsequently thawed and divided into the following three treatment groups: no seminal plasma addition after semen thawing (NOSP); the addition of homologous seminal plasma after semen thawing (HSP) and the addition of autologous seminal plasma after semen thawing (ASP). The addition of 20% of seminal plasma led to an increase in the cell population that simultaneously show plasma and acrosomal membrane integrity (p < 0.05). The addition of seminal plasma did not alter the total motility, the amount of cells with mitochondrial membrane potential or the sperm velocities (average path velocity, straight-line velocity and curvilinear velocity). However, the beat/cross-frequency, straightness and linearity were reduced in ASP and HSP groups (p < 0.05). Unexpectedly, the addition of homologous seminal plasma reduced the proportion of cells with progressive motility (p < 0.05) and the addition of autologous seminal plasma reduced the amplitude of the lateral head displacement (p < 0.05). Based on the increase in the cell populations that had the plasma and acrosomal membrane integrity simultaneously identified in this study, we proposed that the addition of seminal plasma (autologous or homologous) into post-thawed semen before insemination could increase semen fertility.

Simultaneous assessment of plasmatic, acrosomal, and mitochondrial membranes in ram sperm by fluorescent probes / Avaliação simultânea das membranas plasmática, acrossomal e mitocondrial de espermatozoides de carneiros por sondas fluorescentes

In this experiment, it was defined a protocol of fluorescent probes combination: propidium iodide (PI), fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA), and JC-1. For this purpose, four ejaculates from three different rams (n=12), all showing motility >80 percent and abnormal morphology <10 percent, were diluted in TALP medium and split into two aliquots. One of the aliquots was flash frozen and thawed in three continuous cycles, to induce damage in cellular membranes and to disturb mitochondrial function. Three treatments were prepared with the following fixed ratios of fresh semen:flash frozen semen: 0:100 (T0), 50:50 (T50), and 100:0 (T100). Samples were stained in the proposal protocol and evaluated by epifluorescence microscopy. For plasmatic membrane integrity, detected by PI probe, it was obtained the equation: v=1.09+0.86X (R²=0.98). The intact acrosome, verified by the FITC-PSA probe, produced the equation: v=2.76+0.92X (R²=0.98). The high mitochondrial membrane potential, marked in red-orange by JC-1, was estimated by the equation: v=1.90+0.90X (R²=0.98). The resulting linear equations demonstrate that this technique is efficient and practical for the simultaneous evaluations of the plasmatic, acrosomal, and mitochondrial membranes in ram spermatozoa.

Neste experimento, foi definida uma combinação de sondas fluorescentes: iodeto de propídio (PI), aglutinina de Pisum sativum conjugada ao isotiocionato de fluoresceína (FITC-PSA) e JC-1. Para esta proposta, quatro ejaculados de três carneiros (n=12), que apresentavam motilidade >80 por cento e alterações morfológicas <10 por cento, foram diluídos em meio TALP e divididos em duas alíquotas. Uma alíquota foi submetida a três ciclos de flash frozen e descongelação, para induzir danos nas membranas celulares e distúrbios na função mitocondrial. Três tratamentos foram preparados com as seguintes proporções preestabelecidas de sêmen fresco: sêmen submetido a flash frozen: 0:100 (T0), 50:50 (T50) e 100:0 (T100). As amostras foram coradas no protocolo proposto e avaliadas por microscopia de epifluorescência. Para integridade de membrana plasmática, detectada pela sonda PI, foi obtida a equação: v=1,09+0,86X (R²=0,98). O acrossomo intacto, verificado pela sonda FITC-PSA, produziu a equação: v=2,76+0,92X (R²=0,98). O alto potencial de membrana mitocondrial, marcada em vermelho-alaranjado pelo JC-1, foi estimado pela equação: v=1,90+0,90X (R²=0,98). As equações lineares resultantes demonstraram que a técnica é eficiente e prática para avaliação simultânea das membranas plasmática, acrossomal e mitocondrial em espermatozoides de carneiros.

Practical techniques for bovine sperm simultaneous fluorimetric assessment of plasma, acrosomal and mitochondrial membranes.

This experiment was performed to develop and validate practical techniques for simultaneous evaluation of the integrity of plasma and acrosomal membranes, as well as mitochondrial function in bovine spermatozoa using associations of fluorescent probes. Four protocols of fluorescent probes association were defined: protocol 1: propidium iodide (PI), fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and rhodamine 123; protocol 2: PI, FITC-PSA and MitoTracker Green FM (MITO); protocol 3: PI, Hoechst 33342 (H342), FITC-PSA and CMXRos; and protocol 4: PI, H342, FITC-PSA and JC-1. Three ejaculates from each of the four bulls (n = 12) were utilized, showing sperm motility >/=80% and abnormal morphology

Fluorescent stain method for the simultaneous determination of mitochondrial potential and integrity of plasma and acrosomal membranes in boar sperm.

The purpose of this study was to validate a technique for simultaneous evaluation of the plasma, acrosomal and mitochondrial membranes in boar spermatozoa, using an association of fluorescent probes: Propidium iodide (PI), fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and JC-1. Three ejaculates from each of four different boars, all showing motility >or=80% and abnormal morphology