Liquid-stored Semen Research Articles

Fertility after photodynamic inactivation of bacteria in extended boar semen.

Antimicrobial resistance is an increasing challenge in semen preservation of breeding animals, especially in the porcine species. Bacteria are a natural component of semen, and their growth should be inhibited to protect sperm fertilizing capacity and the female's health. In pig breeding, where semen is routinely stored at 17°C in the liquid state, alternatives to conventional antibiotics are urgently needed. Photodynamic inactivation (PDI) of bacteria is a well-established tool in medicine and the food industry but this technology has not been widely adopted in semen preservation. The specific challenge in this setting is to selectively inactivate bacteria while maintaining sperm integrity and functionality. The aim of this study was to test the principle of PDI in liquid stored boar semen using the photosensitizer 5,10,15,20-tetrakis(N-methyl-4-pyridyl)-21H,23H-porphine (TMPyP) and a white light LED-setup. In the first step, photophysical experiments comprising singlet oxygen phosphorescence kinetics of TMPyP and determination of the photosensitizer triplet time revealed a sufficiently high production of reactive singlet oxygen in the Androstar Premium semen extender, whereas seminal plasma acted as strong quencher. In vitro experiments with extended boar semen showed that the established PDI protocol preserves sperm motility, membrane integrity, DNA integrity, and mitochondrial activity while efficiently reducing the bacteria below the detection limit. A proof-of-concept insemination study confirmed the in vivo fertility of semen after photodynamic treatment. In conclusion, using the PDI approach, an innovative tool was established that efficiently controls bacteria growth in extended boar and maintains sperm fertility. This could be a promising contribution to the One Health concept with the potential to reduce antimicrobial resistance in animal husbandry.

Effects of liquid storage of buck semen at refrigeration temperatures on sperm viability and fertility to develop ready to use goat semen diluent

It is generally acknowledged that lower pH throughout liquid storage can decrease sperm motility and energy production, which may be advantageous for maintaining sperm viability during cryopreservation. Hence, the purpose of this study was to see how pH affected sperm viability, motility, membrane integrity, and oxidative changes after 5 days of storage in chemically specified extenders at refrigerator temperatures to formulate a ready-to-use goat semen diluent. Ejaculates were extended with tris-citric acid-fructose diluent with 20% or without egg yolk and stored at 5°C for 5 days. Sperm motility, live count, acrosome integrity, hypo-osmotic swelling positive spermatozoa, pH, and malondialdehyde all substantially differed significantly (P < 0.05) during different days of liquid storage. The pH of diluted semen decreased considerably (P < 0.05) from its original pH of 6.46 on 0 day to pH 6.06 on the 5th day of liquid storage at refrigeration temperature. Diluted semen at refrigeration temperature having 20% egg yolk had significantly higher (P < 0.05) seminal characteristics compared to liquid semen without egg yolk. The liquid storage of semen quality became worse day by day due to a drop in pH and egg yolks acted as a cryoprotectant against the harmful effects of chilling. Hence, 20% egg yolk may be used to enhance the keeping quality of diluted buck semen at refrigeration temperature for up to 5 days. Based on this result, we formulated a ready to use goat semen diluent for liquid semen storage at room temperature and refrigeration temperature.

Phenylbutyrate and Dichloroacetate Enhance the Liquid-Stored Boar Sperm Quality via PDK1 and PDK3

Artificial insemination (AI) with liquid-stored semen is the most prevalent and efficient assisted reproduction technique in the modern pork industry. Pyruvate dehydrogenase complex component X (PDHX) was demonstrated to be associated with sperm metabolism and affected the boar sperm viability, motility, and fertility. Pyruvate Dehydrogenase Kinases (PDKs) are the key metabolic enzymes that regulate pyruvate dehydrogenase complex (PDHC) activity and also the conversion from glycolysis to oxidative phosphorylation. In the present study, two PDK inhibitors, Dichloroacetate (DCA) and Phenylbutyrate (4-PBA), were added to an extender and investigated to determine their regulatory roles in liquid-stored boar sperm at 17 °C. The results indicated that PDK1 and PDK3 were predominantly located at the head and flagella of the boar sperm. The addition of 2 mM DCA and 0.5 mM 4-PBA significantly enhanced the sperm motility, plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), and ATP content. In addition, DCA and 4-PBA exerted their effects by inhibiting PDK1 and PDK3, respectively. In conclusion, DCA and 4-PBA were found to regulate the boar sperm metabolic activities via PDK1 and PDK3. These both can improve the quality parameters of liquid-stored boar sperm, which will help to improve and optimize liquid-stored boar semen after their addition in the extender.

Seasonal stress on semen quality profiles, seminal biochemical and oxidative stress attributes in endangered Teressa goat of Andaman and Nicobar Islands

Objective: To measure seasonal effects on semen quality profiles, seminal biochemical and oxidative stress attributes in fresh and liquid stored semen in monsoon and dry seasons. Methods: A total of 10 Teressa bucks (3-4 years) were selected from breeding farm, ICAR-Central Island Agricultural Research Institute, Port Blair, Andaman and Nicobar Islands, India. Semen samples (n=25 per season) were collected through artificial vagina method and preserved at refrigerated temperature (5°C) for 48 h using Tris citrate glucose based extender. We detected semen quality parameters [volume, mass activity, pH, sperm concentration, total motility, viability, total sperm abnormality, and plasma membrane, acrosomal and nuclear integrities], biochemical profiles [aspartate amino transferase (AST), alanine amino transferase (ALT) and total cholesterol], and oxidative stress markers [total antioxidant capacity (TAC) and malondialdehyde (MDA)] during monsoon and dry seasons. Results: Semen quality parameters significantly differed between seasons (P<0.05) and among storage periods (P<0.05). Volume, pH, mass activity, motility, viability, acrosomal, plasma membrane and nuclear integrities, and TAC were significantly higher (P<0.05). Sperm concentration, sperm abnormalities, MDA, AST, ALT and total cholesterol were significantly lower in fresh semen of monsoon than dry season (P<0.05). Motility, viability, acrosomal, plasma membrane and nuclear integrities, and TAC were significantly decreased (P<0.05) while sperm abnormality, AST, ALT, total cholesterol and MDA were significantly increased as liquid semen storage period advanced (P<0.05). Conclusions: Monsoon season has higher beneficial effects on semen quality profiles and liquid stored semen remained usable for upto 48 h. Good quality ejaculates with higher TAC and lower MDA can be cryopreserved and will be used for artificial insemination.

Spermatozoa quality of Landrace Boar in Beltsville thawing solution diluent with various levels of moringa leaf extract

This study aims to find the best level of sun-dried Moringa leaf extract (SDMLE) in Beltsville Thawing Solution (BTS) diluent to preserve the spermatozoa of Landrace boars. Semen from a two-year-old Landrace boar was collected using the massage method. Semen with spermatozoa motility ≥70% and spermatozoa abnormalities <20% was diluted with BTS (P0, control), BTS + 3% SDMLE (P1), BTS + 6% SDMLE (P2), BTS + 9% SDMLE (P3), and BTS + 12% SDMLE (P4). Storage of liquid semen using a cool box with a temperature of 15-20oC, while evaluating the quality of semen every 8 hours for spermatozoa motility, viability, abnormality, and longevity. The results showed that spermatozoa stored for 40 hours in BTS + 3% SDMLE (P1) diluent had higher quality (P<0.05) than the other four diluents, namely motility (54.00%), viability (61.16%), abnormality (3.62%), and longevity (45.77%). The results of this study concluded that 3% SDMLE added to BTS diluent was the best level for the preservation of spermatozoa in landrace boars.

Vaginal and vestibular electrical resistance as an alternative marker for optimum timing of artificial insemination with liquid-stored and frozen-thawed spermatozoa in sows

Development of a pen-side test to objectively determine the ideal time for artificial insemination (AI) in the sow would save producers time and money. Current processes rely on identification of oestrus via subjective behavioural and physiological markers that are indicative of high blood oestrogen concentrations. This study attempted to use measurements of electrical resistance (ER) in the cervical mucus to pinpoint timing of AI accurately enough to lead to equivalent pregnancy rates as a natural mating. Thirty-six sows were divided into 3 groups and observed for signs of oestrus. Seven sows did not show any oestrus behaviour and were excluded from the study. The remaining 29 sows were inseminated via natural mating and conventional oestrus detection (n = 14), or inseminated artificially with either liquid-stored semen (n = 8) or frozen-thawed semen (n = 7) according to timing indicated from electrical resistance measurements in the vagina and vestibule. Sows that were artificially inseminated on the basis of the electrical resistance readings had a lower pregnancy rate (P = 0.034) and less piglets born alive per litter (P < 0.05) than those that were naturally mated according to a conventional oestrus detection regime. However, the pregnancy rate and total piglets born alive did not differ between the two groups that underwent artificial insemination. Change in electrical resistance in the vagina has the potential to accurately predict ovulation timing, but more work is required to refine the timing of AI in relation to the readings before the technique can be adopted by industry.

Analysis of changes in the morphological structures of sperm during preservation of liquid boar semen in two different seasons of the year

Animal fertility is the result of a combination of genetic, physiological and environmental factors. Assessment of semen quality plays a key role in determining the reproductive performance of boars. The aim of the study was to determine the effect of two seasons of the year on the morphology, morphometry, cell membrane integrity and mitochondrial activity of sperm during storage of liquid boar semen at 17 °C. The study was carried out using 20 boars. Four ejaculates were collected from each boar in each of two seasons of the year. All tests were carried out five times: at 1, 24, 48, 96 and 168 h during storage. The results showed that sperm in ejaculates collected in summer are more sensitive to storage conditions than those from ejaculates obtained in winter. The percentage of sperm with morphological defects was shown to increase with the storage time of the diluted ejaculates, particularly between 96 and 168 h of preservation. In summer, the percentage of sperm with an intact cell membrane and the percentage with high mitochondrial membrane potential are much lower than in winter, at every hour of semen preservation. In the case of boars used for artificial insemination, it is worth taking into account the season when the semen is collected, especially if it is to be stored in liquid form. Assessment of sperm cell structures during storage of liquid semen should be implemented at insemination stations and should be carried out more often in summer.

Effects of Using Commercial and Homemade Extenders on Sperm Quality of Liquid Stored Semen of Horro Chicken Breed

This study aimed to evaluate the suitability of homemade tris-egg yolk-based and Commercial Beltsville poultry extenders for short-term storage of semen from the Ethiopian Indigenous Horro chicken breed at refrigeration temperature. A total of 30 Horro roosters with an average age of 40 weeks were used to collect semen. The treatments (T) in the sperm quality experiment were control (semen without extender added), semen extended with homemade extender (E1), and semen extended with commercial Beltsville Poultry Semen Extender (E2). Changes in spermatozoa motility, in vitro viability, and morphology were evaluated in fresh semen and semen diluted as 1:4 (v/v semen to extender) and stored for 4, 8, 12, and 24 hours at 4°C. During semen storage, there was a decrease in mass motility, an increase in morphologically abnormal spermatozoa with a high incidence of the bent tail, and an increase in dead spermatozoa. The commercial Beltsville poultry extender was found to be the most suitable extender regarding mass motility and in vitro viability of stored spermatozoa, but there was no significant difference in sperm abnormalities across all extenders. The results showed locally prepared tris-egg yolk-based extender could be a suitable extender for short-term storage of chicken sperm regarding the sperm quality attributes.

Animal board invited review: An update on the methods for semen quality evaluation in swine - from farm to the lab.

Pig breeding is mainly conducted through artificial insemination with liquid-stored semen. It is, therefore, crucial to ensure that sperm quality is over the standard thresholds, as reduced sperm motility, morphology or plasma membrane integrity are associated with reduced farrowing rates and litter sizes. This work aims to summarise the methods utilised in farms and research laboratories to evaluate sperm quality in pigs. The conventional spermiogram consists in the assessment of sperm concentration, motility and morphology, which are the most estimated variables in farms. Yet, while the determination of these sperm parameters is enough for farms to prepare seminal doses, other tests, usually carried out in specialised laboratories, may be required when boar studs exhibit a decreased reproductive performance. These methods include the evaluation of functional sperm parameters, such as plasma membrane integrity and fluidity, intracellular levels of calcium and reactive oxygen species, mitochondrial activity, and acrosome integrity, using fluorescent probes and flow cytometry. Furthermore, sperm chromatin condensation and DNA integrity, despite not being routinely assessed, may also help determine the causes of reduced fertilising capacity. Sperm DNA integrity can be evaluated through direct (Comet, transferase deoxynucleotide nick end labelling (TUNEL) and its in situ nick variant) or indirect tests (Sperm Chromatin Structure Assay, Sperm Chromatin Dispersion Test), whereas chromatin condensation can be determined with Chromomycin A3. Considering the high degree of chromatin packaging in pig sperm, which only have protamine 1, growing evidence suggests that complete decondensation of that chromatin is needed before DNA fragmentation through TUNEL or Comet can be examined.

Effect of genotypes on reaction time, refractory period, semen index and liquid stored semen quality of breeding bulls in Bangladesh

The present study was conducted with the aim to reveal the effect of genotypes on reaction time, refractory period, semen index and liquid stored (at 4oC) semen quality of breeding bulls in Bangladesh. Semen was collected twice a week from Holstein Friesian, Sahiwal and Brahman breeding bulls. Reaction time and refractory period were examined for the breeding bulls. Semen volume, sperm concentration and total spermatozoa/ejaculate were measured immediately after collection. It was revealed that genotype had a significant (P&lt;0.01) effect on semen volume, sperm concentration, total sperm output and refractory period but not in reaction time. From the point of view of semen index, semen quality of Holstein Friesian breeding bulls was superior to Sahiwal and Brahnman breeding bulls. Progressive motility, live and normal spermatozoa of fresh semen did not differ significantly but after dilution progressive motility differed significantly (P&lt;0.05) in different genotypes of breeding bulls. During preservation time (0 to 120 hours), progressive motility, normal and live spermatozoa changed significantly (P&lt;0.05) in each genotype with the progress of time. During 0 hour, 72 hours and 120 hours of preservation, progressive motility was found in Holstein Friesian (73.11±1.12%, 53.11±5.14%, 13.40±2.53%), Sahiwal (78.21±1.68%, 64.96±4.60%, 13.22±1.42%), and Brahman (75.21±1.68%, 54.86±4.40%, 16.96±4.42%), respectively. On the other hand, non-return rate was found insignificant (P&gt;0.05), where higher fertility was observed in Holstein Friesian bull (67.2%) followed by Sahiwal (63.7%) and Brahman bull (57.38%). In a nutshell, Holstein Friesian bull has better fresh, diluted and preserved semen quality than the other two genotypes and after the 3 days of preservation, semen quality in respect of progressive motility, normal and live spermatozoa drastically deteriorated. Therefore, it is recommended that preserved semen of different genotypes should be used for AI within 3 days of liquid semen preservation at 4oC.

Extender ability to Store Chicken Sperm by using L-Carnitine, Methionine and Honey

This study was carried out to investigate the effect of L-Carnitine (LC), L-Methionine (LM) and Honey (H) supplementation on Lake’s diluent ability to store chicken sperm. Semen samples were collected from local meat type chicken males (n=20, by abdominal massage method. Two experiments were carried out. In Exp-I twelve treatments were prepared in Lake’s diluent mixing different concentrations of LC (i.e., 0.5mMLC, 1mMLC, 2mMLC and 4mMLC), LM (i.e., 0.5mMLM, 1mMLM, 2mMLM and 4mMLM) and H (i.e., 0.25%H, 0.5%H, 1%H and 2%H) while Lake’s diluent without any addition was used as control. In Exp-II four treatments were prepared by mixing different proportions of LC + LM + H in Lake’s diluent as followed: T1 (0.125mMLC + 0.125LM + 0.125%H), T2 (0.25mMLC + 0.25LM + 0.25%H), T3 (0.5mMLC + 0.5LM + 0.5%H) and T4 (1mMLC + 1mMLM + 1%H), while semen mixed only with Lake’s diluent was used as control (To). In both experiments semen was stored up till 48h at 5°C and sperm motility, viability, membrane integrity and abnormal morphology were assessed at 0, 12, 24 and 48h of storage. In Exp-I after 48h we recorded highest (P&lt;0.05) motility (62%) with 2mMLM, membrane integrity with 0.25%H and 0.5%H (43%), sperm viability with 0.25% H (70%) compared with all other treatments, while lowest morphological defects with 1mMLC (29%) and 1mMLM (29%). In Exp-II at 48h we observed highest (P&lt;0.05) motility (52%) and membrane integrity (48%) with T1 compared with all other treatments. We observed highest (P&lt;0.05) sperm viability (64%) with T2 while T1 and T2 had lowest morphological defects (Avg. 29%). It is concluded that 2mMLM supports high motility while honey support viability and membrane integrity till 48h. In addition to that a combination of 0.125mMLC + 0.125mMLM + 0.125%H gives good protection to sperm during liquid semen storage in Lake’s diluemt.

Effects of water-soluble curcuminoid-rich extract in a solid dispersion form (CRE-SD) on the sperm characteristics, longevity and casein kinase II catalytic subunit alpha protein stability in chilled goat semen

The present study evaluated the effects of water-soluble curcuminoid-rich extract in a solid dispersion form (CRE-SD) on goat sperm qualities and sperm protein CSNK2A2 expression during liquid storage. Semen was collected from five fertile goats, using an artificial vagina. Ejaculates with a motility above 70% were cooled to 4 °C using TRIS-citric acid-fructose diluent with 10% egg yolk containing various concentrations of CRE-SD (0, 0.1, 1, 10 and 100 μg/mL). Chilled sperm were evaluated for sperm characteristics, casein kinase II catalytic subunit alpha (CSNK2A2) protein level and oxidative status up to 15 days. After 12 days of preservation, sperm motility, viability, acrosomal integrity and mitochondrial activity were significantly higher in the group preserved with 10 μg/mL CRE-SD as compared with the control group. Supplementation of CRE-SD at this concentration was also able to conserve the CSNK2A2 a significantly higher than that in control group until 9 days of cold storage, possibly by reducing oxidative stress. The molecular mass of the sperm CSNK2A2 protein detected in this study was 37 kDa; it was mostly located in the post-acrosomal region, midpiece and flagellum. These results demonstrate the possibility to use the CRE-SD as a natural antioxidant during liquid semen storage in goats.

A 5-color flow cytometry panel to assess plasma membrane integrity, acrosomal status, membrane lipid organization and mitochondrial activity of boar and stallion spermatozoa following liquid semen storage

For a more practically applicable analysis of different sperm characteristics, this study aimed to develop a 5-color flow cytometry (FC) panel to concurrently analyze four sperm parameters in liquid boar and stallion semen, using also a DNA-marker for selecting sperm cell events. From each of thirty extended boar semen doses and twelve stallion semen doses, six aliquots were taken. For evaluating mitochondrial activity (A), degree of lipid disorder of plasma membrane (B), integrity of plasma membrane (C), acrosomal status (D) and marking DNA (E), five aliquots were individually stained with Rhodamine 123, Merocyanine 540, Propidium Iodide, PNA-Alexa Fluor 647, and Hoechst 33342, respectively. The sixth aliquot was stained with all the five fluorochromes simultaneously, whereas spectral overlap was corrected by a compensation matrix. Strong correlations were found between the single and 5-color staining assays for boar sperm (A: 0.99, B: 0.96, C: 0.93, D: 0.98, E: 0.99; P < 0.01). Furthermore, moderate and substantial Concordance Correlation Coefficients (CCC) were presented by all these parameters (0.99, 0.96, 0.92, 0.98, and 0.99, respectively). For stallion sperm, the correlation coefficients between the assays were also strong (A: 0.99, B: 0.98, C: 0.99, D: 0.99, E: 0.95; P < 0.01) and substantial CCC were observed for all of them (0.99, 0.97, 0.99, 0.99, and 0.90, respectively). For both species, the mean difference between the methods (d̅) did not overtake 0.84. The results confirmed that this 5-color panel could be successfully implemented for analyzing boar and stallion sperm quality in a single, practical and quick FC assay.

Effect of sow’s parity and semen storage time on reproductive performance of pig after artificial insemination with liquid boar semen

Artificial insemination with liquid stored boar semen has considerably improved the pig genetic throughout the world. The present study analyzed the effect of sow's parity and semen storage time on reproductive performance of pig after artificial insemination with liquid boar semen. A total of 218 Gunghroo × Hampshire female pigs including gilts (42) and sows (176) were inseminated with liquid stored semen. Reproductive performance parameters, including farrowing rate, gestation length, weaning to estrous interval, total piglets born, total live piglets born, stillborn piglets, mummified piglets and litter size at weaning were analyzed between different parity and also between different semen storage duration (24 h, 24 to 48 h and 48 to 72 h). There was no effect of parity on farrowing rate. Gestation length was significantly higher in third, fourth and fifth parity as compared to first and second parity. Weaning to estrus interval was significantly higher in first parity. Total piglets born and total live piglets born were significantly higher in third, fourth and fifth parity as compared to first and second parity. There was no effect of parity on stillborn and mummified piglets. Litter size at weaning was significantly higher in third to fifth parity as compared to first and second parity. There was no effect of semen storage duration on farrowing rate, gestation length and weaning to estrus interval. Total piglets born, total live piglets born and litter size at weaning were significantly higher in first group as compared to second and third group. There was no effect of semen storage time on stillborn and mummified piglets. In conclusion, artificial insemination in pig could be successfully used with promising reproductive performance under sub-tropical climatic condition. However, a decrease in litter size was observed when insemination was done with semen stored for more than 24 h.

Exogenous gamma-aminobutyric acid addition enhances porcine sperm acrosome reaction.

The widely used porcine artificial insemination procedure involves the use of liquid‐stored semen because it is difficult to control the quality of frozen–thawed porcine sperm. Therefore, there is a high demand for porcine semen. The control and enhancement of sperm function are required for the efficient reproduction of pigs. We previously reported that gamma‐aminobutyric acid (GABA) enhanced sperm capacitation and acrosome reaction in mice. In this study, we demonstrated the presence of GABAA receptors in porcine sperm acrosome. Furthermore, we investigated the GABA effects on porcine sperm function. We did not detect any marked effect of GABA on sperm motility and tyrosine phosphorylation of sperm proteins. However, GABA promoted acrosome reaction, which was suppressed by a selective GABAA receptor antagonist. GABA binds to GABAA receptors, resulting in chloride ion influx. We found that treatment with 1 μM GABA increased the intracellular concentration of chloride ion in the sperm. In addition, the GABA concentration effective in the acrosome reaction was correlated with the porcine sperm concentration. These results indicate that GABA and its receptors can act as modulators of acrosome reaction. This study is the first to report the effects of GABA on porcine sperm function.

Melatonin and canthaxanthin enhances sperm viability and protect ram spermatozoa from oxidative stress during liquid storage at 4°C.

Antioxidants are used to minimize oxidative stress during liquid semen storage. The main aim of current study was to elucidate effect of supplementing melatonin and canthaxanthin in Tris-based extender could enhance seminal quality of ram at 4°C up to 72h. A total of 48 ejaculates were collected from breeding Magra rams (n=8) and were preliminarily subjected for various macroscopic and microscopic semen evaluation tests. These ejaculates were pooled and divided into three equal aliquots. Two aliquots were diluted (1:10) using extender encompassing final concentration of 1mM melatonin and 25µM canthaxanthin and stored at 4°C. Third aliquot with extender only was kept as control. Structural and functional seminal changes were observed at different time points of preservation. Results revealed that mean values for progressive sperm motility, viability and total antioxidant capacity were significantly higher (p<0.05) in melatonin group while hypo-osmotic swelling test was significantly (p<0.05) higher in canthaxanthin group. Total sperm abnormalities and malondialdehyde levels were significantly (p<0.05) lower in both treatment groups indicating their antioxidant efficacy in protection of spermatozoa from oxidative stress. Results of study indicated that supplementation of these antioxidants to ram semen could be used to enhance storage life of liquid semen at 4°C up to 72h.

Oxytocin in pig seminal plasma is positively related with in vivo fertility of inseminated sows

BackgroundIdentification of relevant in vivo biomarkers for fertility remains a challenge for the livestock industry. Concentrations of the small peptide hormone oxytocin (OXT), involved in male reproductive function and present in the seminal plasma (SP) of several species could be a robust one. This study characterized concentrations of SP-OXT in ejaculates from boars used in artificial insemination (AI) programs aiming to evaluate its relationship with sperm quality variables and in vivo fertility of their liquid-stored AI-semen. Seminal OXT concentrations (ng/mL) were measured in 169 ejaculates from 61 boars of the Duroc, Pietrain, Landrace and Large White breeds using a direct competitive immunoassay test based on AlphaLISA® technology. Ejaculate (ejaculate volume, sperm concentration, total sperm count) and sperm parameters (motility, viability, intracellular generation of reactive oxygen species, plasma membrane fluidity) were assessed at 0 h and 72 h in AI-semen samples stored at 17 °C. In vivo fertility included only 18 Large White and Landrace boars whose AI-semen was used to inseminated > 100 sows and evaluated both farrowing rate and litter size of 3,167 sows.ResultsThe results showed that SP-OXT differed between boars and between ejaculates within boar (P < 0.05) but not between breeds (Duroc, Pietrain, Landrace and Large White). Ejaculates with higher SP-OXT concentration/mL (hierarchically grouped; P < 0.001) had larger volume and came from younger boars (P < 0.05). Ejaculates of boars showing positive farrowing rate deviation exhibited higher (P < 0.05) SP-OXT concentration/mL than those with negative farrowing rate deviation.ConclusionThe SP concentrations of OXT are boar, ejaculate and age dependent, and positively related with ejaculate volume and farrowing rates of liquid-stored semen AI-doses.

Monitoring the reactive oxygen species in spermatozoa during liquid storage of boar semen and its correlation with sperm motility, free thiol content and seasonality.

Oxidative stress is an important factor affecting the quality of spermatozoa during liquid storage of boar semen; however, monitoring of reactive oxygen species (ROS) that provides direct insight into the oxidative status is not yet attempted. This study aimed to monitor ROS in boar sperm during liquid semen storage to determine its correlation with sperm motility and free thiol (SH) content, and seasonality. Ejaculate was collected from mature Duroc boars in a commercial farm in autumn and spring, diluted in Mulberry III extender, stored at 15°C, and examined daily for sperm ROS level, SH content and motility. The ROS levels in spermatozoa prepared during autumn and spring were constantly low until days 4 and 5 of storage, respectively, which thereafter progressively increased in association with the loss of sperm motility. The increased sperm ROS level correlated with the higher SH level and lower motility, which was accentuated from day 4 of storage and was higher in September, or early autumn. This study indicates that increased sperm ROS levels during liquid storage results in oxidative damage, causing loss of sperm motility, presumably through decreased sperm viability, suggesting that sperm ROS monitoring effectively evaluates the quality of boar semen.

Seminal Plasma Anti-Müllerian Hormone: A Potential AI-Boar Fertility Biomarker?

The anti-Müllerian hormone (AMH), a Sertoli cell-secreted glycoprotein that is present in seminal plasma (SP), is considered as a marker of spermatogenesis in humans. This study aimed to evaluate the presence of this hormone in boar SP, together with its putative relationship with sperm quality, function, and in vivo fertility parameters in liquid-stored semen samples. The concentration of SP-AMH was assessed in 126 ejaculates from artificial insemination (AI)-boars (n = 92) while using a commercial Enzyme-Linked ImmunoSorbent Assay (ELISA) kit with monoclonal antibodies specific for Sus scrofa AMH (CEA228Po, Cloud-clone). Sperm quality (concentration, motility, viability, and acrosome damage) and functionality (membrane lipid disorder and intracellular H2O2 generation) were assessed in semen samples at 0 and 72 h of liquid-storage. In addition, fertility parameters from 3113 sows inseminated with the AI-boars were recorded in terms of farrowing rate, litter size, number of stillbirths per litter, and the duration of pregnancy over a 12-month period. The results revealed that the SP-AMH concentration varied widely among boar ejaculates, with no differences among breeds. Moreover, the SP-AMH concentration proved to be a good predictive biomarker for sperm concentration (p ˂ 0.05), but poor for other sperm quality, functionality, and in vivo fertility parameters of liquid-stored semen samples from AI-boars.

Glutathione S-Transferases Play a Crucial Role in Mitochondrial Function, Plasma Membrane Stability and Oxidative Regulation of Mammalian Sperm.

Glutathione S-transferases (GSTs) are essential sperm antioxidant enzymes involved in cell protection against oxidative stress and toxic chemicals, preserving sperm function and fertilising ability. Artificial insemination (AI) in pigs is commonly carried out through the use of liquid-stored semen at 17 °C, which not only reduces sperm metabolic activity but also sperm quality and AI-farrowing rates within the 72 h of storage. While one may reasonably suggest that such enzymes are implicated in the physiology and maintenance of mammalian sperm function during liquid-storage, no previous studies conducted on any species have addressed this hypothesis. Therefore, the objective of the present work was to characterise the presence and function of sperm GSTs in mammalian sperm, using the pig as a model. In this regard, inhibition of such enzymes by ethacrynic acid (EA) during semen storage at 17 °C was performed to evaluate the effects of GSTs in liquid-preserved boar sperm by flow cytometry, immunofluorescence, and immunoblotting analysis. The results of this study have shown, for the first time in mammalian species, that the inhibition of GSTs reduces sperm quality and functionality parameters during their storage at 17 °C. These findings highlight the key role of such enzymes, especially preserving mitochondrial function and maintaining plasma membrane stability. In addition, this study has identified and localised GSTM3 in the tail and equatorial subdomain of the head of boar sperm. Finally, this study has set grounds for future investigations testing supplementation of semen extenders with GSTs, as this may improve fertility outcomes of swine AIs.