Sperm-rich Fraction Research Articles

Nerve Growth Factor-β production in the bull: Gene expression, immunolocalization, seminal plasma constitution, and association with sire conception rates

Nerve Growth Factor-β (NGF) is a seminal plasma protein that regulates sperm physiology in bulls, yet its production and association with fertility remain unclear. The objective of this study was to describe NGF distribution in bull accessory sex glands, quantify its seminal plasma concentrations, and determine its association with sire conception rates. Accessory sex glands were collected post-mortem from mature bulls (n = 3). Quantitative PCR and immunohistochemistry were performed in tissue samples. Pre-ejaculate and sperm-rich fraction of seminal plasma from Angus (n = 42) and sperm-rich fraction from Holstein (n = 10) bulls were collected by electroejaculation. Holstein (n = 76) and Jersey (n = 14) bulls were collected by artificial vagina, and those with available sire conception rate scores were allocated to: (1) negative/0 (n = 15); or (2) positive (n = 45). Seminal plasma NGF concentrations were measured using ELISA. Relative abundance of NGF mRNA was greatest in the vesicular, intermediate in ampulla, and lowest in prostate and bulbourethral glands (P ≤ 0.03). The most intense NGF staining was detected in the ampulla and vesicular glands (P < 0.05). Concentrations of NGF were greater in the sperm-rich fraction than in the pre-ejaculate and in samples collected by artificial vagina (P < 0.01). Seminal plasma NGF concentrations were greater in bulls with positive sire conception rate than those with negative/0 (P = 0.05). This study determined that NGF is produced in the ampulla and vesicular glands of bulls and is secreted into the sperm-rich fraction of the ejaculate. The positive association of NGF with bull fertility warrants further investigation.

Cryopreservation of boar semen in 0.5mL straws at low spermatozoa concentration is better than high concentration to maintain sperm viability

ABSTRACT: To date, no studies have been performed evaluating the effect of boar spermatozoa concentration in 0.5mL freezing straws, leading us to examine this question. Each sperm-rich fraction of the ejaculate (n=25) was diluted at five different sperm concentrations (100, 200, 300, 600 and 800 x 106 spermatozoa/mL), packaged in 0.5mL straws, and subsequently frozen. After thawing, the sperm from all of treatment groups were analyzed to determine motility characteristics using a sperm class analyzer (SCA-CASA), and their plasma and acrosomal membrane integrity, mitochondrial membrane potential, sperm membrane lipid peroxidation and fluidity were analyzed by flow cytometry. An increase in spermatozoa concentration above 300x106 spermatozoa/mL in a 0.5mL straw impaired (p&lt;0.05) the total and progressive motility, curvilinear velocity, straight-line velocity, linearity and beat cross frequency. However, the plasma and acrosomal membrane integrity, mitochondrial membrane potential, membrane lipid peroxidation and fluidity were not influenced (p&gt;0.05) by high spermatozoa concentrations at freezing. Therefore, to increase spermatozoa survival and total and progressive motility after thawing, boar spermatozoa should be frozen at concentrations up to 300x106 spermatozoa/mL.

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.

Breeding soundness evaluation (BSE) in boars from Costa Rica

Nerve Growth Factor-β (NGF) is a seminal plasma protein that regulates sperm physiology in bulls, yet its production and association with fertility remain unclear. The objective of this study was to describe NGF distribution in bull accessory sex glands, quantify its seminal plasma concentrations, and determine its association with sire conception rates. Accessory sex glands were collected post-mortem from mature bulls (n = 3). Quantitative PCR and immunohistochemistry were performed in tissue samples. Pre-ejaculate and sperm-rich fraction of seminal plasma from Angus (n = 42) and sperm-rich fraction from Holstein (n = 10) bulls were collected by electroejaculation. Holstein (n = 76) and Jersey (n = 14) bulls were collected by artificial vagina, and those with available sire conception rate scores were allocated to: (1) negative/0 (n = 15); or (2) positive (n = 45). Seminal plasma NGF concentrations were measured using ELISA. Relative abundance of NGF mRNA was greatest in the vesicular, intermediate in ampulla, and lowest in prostate and bulbourethral glands (P ≤ 0.03). The most intense NGF staining was detected in the ampulla and vesicular glands (P < 0.05). Concentrations of NGF were greater in the sperm-rich fraction than in the pre-ejaculate and in samples collected by artificial vagina (P < 0.01). Seminal plasma NGF concentrations were greater in bulls with positive sire conception rate than those with negative/0 (P = 0.05). This study determined that NGF is produced in the ampulla and vesicular glands of bulls and is secreted into the sperm-rich fraction of the ejaculate. The positive association of NGF with bull fertility warrants further investigation.

A comparative study of effects of different freezing methods on sperm quality, DNA integrity and membrane protein of cryopreserved boar semen*

The present study determined the effects of conventional, and controlled freezing method adopting three freezing rates, viz. 20°C, 40°C and 60°C/min on quality of sperm (motility, viability and plasma membrane integrity), DNA integrity and plasma membrane protein profile of cryopreserved boar semen. Sixty sperm-rich fractions of ejaculates from six boars were utilized for freezing of semen with different freezing methods in lactose-egg yolk glycerol extender. Semen samples were evaluated for sperm motility, viability (Propidium Iodide assay), functional integrity of plasma membrane (HOST), DNA integrity (Acridine Orange stain) and plasma membrane protein profile (SDSPAGE) after equilibration and after freezing. The results revealed that the post thaw sperm motility, sperm viability, and plasma membrane integrity (HOST-reacted) were significantly higher in all the three controlled freezing methods (20°C, 40°C and 60°C/min) as compared to that in conventional method. In addition, the number of sperm plasma membrane protein loss was less in controlled freezing methods as compared to that in conventional freezing. However, the post thaw sperm DNA integrity did not influence by difference in freezing methods. No significant difference on the post thaw sperm characteristics was recorded among the three controlled freezing rates. All the sperm parameters assessed declined significantly after freezing as compared to that after equilibration irrespective of freezing method employed. In conclusion, controlled freezing methods conferred better post thaw sperm quality as compared to conventional method, and thus the freezing rates of either 20, 40 or 60°C/min could provide better freezability of boar semen.

Evidence of porcine epidemic diarrhea virus (PEDV) shedding in semen from infected specific pathogen-free boars

In 2013, PED emerged for the first time in the United States (US). The porcine epidemic diarrhea virus (PEDV) spread quickly throughout North America. Infection with PEDV causes watery diarrhea and up to 100% mortality in piglets, particularly for highly pathogenic non-InDel strains circulating in the US. PEDV is mainly transmitted by the fecal–oral route. Transmission via the venereal route has been suspected but not previously investigated. The aim of the study was to determine if PEDV could be detected in semen from infected specific pathogen-free (SPF) boars inoculated with a PEDV US non-InDel strain suggesting venereal transmission may occur. Two boars orally inoculated with PEDV showed clinical signs and virus shedding in feces. Transient presence of the PEDV genome was detected by RT-qPCR in the seminal (5.06 × 102 to 2.44 × 103 genomic copies/mL) and sperm-rich fraction of semen (5.64 × 102 to 3.40 × 104 genomic copies/mL) and a longer duration of viral shedding was observed in the sperm-rich fraction. The evidence of PEDV shedding in semen raises new questions in term of disease spread within the pig population with the use of potentially contaminated semen.

Effect of ostrich egg lipoproteins and hen egg yolk on the quality of dog sperm during liquid storage at 5°C

Abstract The objective of this study was a comparative analysis of the impact of the addition of lipoproteins from ostrich egg yolk (LPFo) and hen egg yolk (HEY) to a Tris-citric acid-fructose (TCF) extender on the quality of dog sperm preserved at 5°C for 7 days. The sperm-rich fraction of the ejaculate was manually collected and extended using the following extenders: control (TCF), TCF+ 5% addition of LPFo (TCF-LPFo) and TCF+ 20% of addition HEY (TCF-HEY). The analysis of quality included: sperm motility parameters (TMOT, PMOT, VAP, VSL, VCL, ALH, BCF, STR, LIN), normal apical ridge acrosome (NAR), plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), and ATP content. The addition of LPFo and HEY to the TCF extender had a beneficial effect on all quality parameters of the stored sperm compared with the TCF extender. However, the analysis of sperm motility parameters revealed differences (P≤0.05) between TCF-LPFo and TCF-HEY extenders as affected by storage day. Semen extended in TCF-LPFo was characterized by lower values of TMOT and VAP on Day 5 and by lower values of TMOT and VCL on Day 7, compared to that extended in TCF-HEY. The analysis of PMOT, VSL, ALH, BCF, STR, LIN, PMI, MMP and ATP content showed no differences (P≥0.05) between TCF-LPFo and TCF-HEY extenders. The results suggest that TCF extender supplementation with LPFo or HEY improves the quality of dog sperm stored at 5°C. Both LPFo and HEY protect motility, membrane integrity and can contribute to the improvement of the energy status of stored dog sperm. LPFo can be alternatively used instead of HEY for dog semen preservation in the liquid state.

94 Semen Parameters from a Captive Population of the Endangered Chacoan Peccary (Catagonus wagneri) in Paraguay

Priority conservation actions for the endangered Chacoan peccary (Catagonus wagneri) include improvement of captive breeding initiatives. Eleven captive male peccaries housed in mixed-sex groups of 17 to 35 animals at the Centro Chaqueño para la Conservación e Investigación in Paraguay were immobilised with intramuscular tiletamine/zolazepam (30 mg; Zoletil® 50, Virbac, Forth Worth, TX, USA), azaperone (10 mg; 50 mg mL−1; ZooPharm, Windsor, CO, USA), and medetomidine (1 mg;10 mg mL−1; ZooPharm). Animals were administered supplemental intranasal oxygen (1-2 L min−1) and reversed with atipamazole (5 mg; 25 mg mL−1; ZooPharm). Testis length, width, and firmness (1-3 scale; 1 = hard, 2 = normal, 3 = flaccid) were recorded. The preputial sheath was clipped, cleaned, and flushed with sterile saline using a Foley catheter. A lubricated 13-mm-diameter electroejaculator probe (Beltron Instruments, Longmont, CO, USA) was inserted into the rectum to a depth of 5 cm. The electroejaculation protocol consisted of 4 cycles of 10 stimuli each (1-4 V/cycle). Collection tubes were changed frequently during electroejaculation to avoid urine or gel fraction contamination of the sperm-rich fraction. Collected semen was evaluated for volume, color/appearance, and pH. Percent motility was determined as the average motility of spermatozoa from 10 fields (40×). Progressive motility of spermatozoa was scored on a scale of 0-5 (0 = dead sperm; 5 = rapid forward progression). Semen samples were assessed for total spermatozoa. Percent of live spermatozoa and spermatozoa morphology were evaluated with Eosin-Negrosin smears. The mean ± standard error of the mean of each parameter is presented. A Pearson correlation coefficient between age and semen parameters was calculated. Males assessed in this study were 10.5 ± 1.2 years old. The mean testis volume was 1005.7 ± 50.9 cm3 and mean testis firmness was 2.1 ± 0.1. The mean ejaculate volume was 2.9 ± 0.7 mL and pH was 7.7 ± 0.3. Few males had motile spermatozoa (n = 5/9), which yielded a mean percent motility of 18.3 ± 8.5 (range: 0-50%) and a mean progressive motility of 0.6 ± 0.3 (range: 0-2). The mean percentage of live spermatozoa was 25.1 ± 5.6 (range: 4-44%). Male Chacoan peccaries in this population had a low percentage of normal spermatozoa (12.4 ± 2.5%; range: 2.8-24.0%). The mean total count of spermatozoa per ejaculate was 1.58 ± 1.01 million (range: 0.09-8.28 million). Spermatozoa defects were predominantly primary (67.1%), with the most common defects being tapered head (15.4 ± 6.2%), diadem/crater (14.8 ± 2.6%), and excess residual cytoplasm (8.5 ± 2.7%). There was no correlation between age and semen parameters (percent live: r = –0.19; motility percentage: r = 0.01; percent normal spermatozoa: r = 0.38; total count: r = 0.29; P &gt; 0.05). This is the first report of semen parameters in the Chacoan peccary. It is unknown whether the poor semen quality is inherent to the species or this population. Reproductive assessment of males from other captive populations and of wild male Chacoan peccaries is warranted.

Effect of sorting boar spermatozoa by sex chromosomes on oviduct cell binding

This study examined the hypothesis that flow sorting sperm by sex chromosomes affects oviduct cell binding which would influence formation of the sperm reservoir in the oviduct. The sperm-rich fraction from boars (n = 5) was collected, sperm were stained with Hoechst 33342 and sorted. Sperm were sorted based on the presence of either an X or Y chromosome and placed into the following treatments: 1) sperm selected for the Y chromosome, 2) selected for the X, 3) an equal mixture of sorted X and Y, and 4) a control of non-sorted sperm from the same collection. Samples were tested for oviduct cell binding within 12 h of sorting. Additionally, sperm were analyzed for motility characteristics, acrosome status, and binding to the two oviduct glycan motifs that bind porcine sperm, biantennary 6-sialylated N-acetyllactosamine on a mannose core (bi-SiaLN) and sulfated LeX trisaccharide (suLeX). The disaccharide found within both glycan motifs, N-acetyllactosamine (LacNAc), was used as a control. Sperm binding to oviduct cells was reduced by more than half in the three sorted samples when compared to the control sperm that were not sorted. The percentage of sperm that were motile 24 h after sorting was also decreased significantly in each of the sorted sample groups when compared to the unsorted control. In contrast, sorting did not decrease the percentage of sperm that bound purified soluble glycans or the location on sperm to which they bound. There was also no difference in sperm acrosome status among the four groups. In summary, sorting reduced sperm binding to the complex matrix around oviductal cell aggregates but sperm binding to purified soluble oviduct glycans was not affected. The requirement for higher affinity and motility to bind glycans immobilized on oviduct cells may explain this difference. The reduction in sperm fertility observed following sex-sorting may be explained partially by a reduced or altered ability to bind to the oviduct epithelium.

Induction of immune-related gene expression by seminal exosomes in the porcine endometrium

Seminal plasma (SP) is considered as a vehicle to carry sperm into female reproductive tract, of which functions have not been completely understood. This study aimed to identify the function of seminal exosomes on porcine endometrium. Exosomes were isolated from the sperm-rich fraction of boar semen and were confirmed by the expression of exosome marker HSP70 and size distribution using nano-sight tracking analysis. Porcine endometrial epithelial cells (EECs) were then treated with seminal exosomes, and RNA extracted were subjected to global expression analysis. Transcripts related to “immune response”, “inflammatory response” and their associated signaling pathways were up-regulated in EECs treated with seminal exosome, whereas those associated with “steroid biosynthesis”, “metabolic pathways” and “T cell differentiation” were down-regulated. The decrease in PMVK, SC5D, INSIG1, HSD17B7, NSDHL, HMGCR, SQLE and FDFT1, and increase in CCL20, TNFSF15, AMCFII, CXCL2 and CXCL8 were also found in the endometrium from the naturally mated pigs. Moreover, changes in exosome-induced CYP24A1, EBP, CCL20, AMCFII and IL1A expression were not regulated by the exosome removed SP. These observations indicated that exosomes present in SP are involved in the immune-related gene regulation in the uterus, which could pave the passage for sperm and possibly fertilized eggs.

Seminal plasma antioxidants are directly involved in boar sperm cryotolerance

Boar ejaculates are ejected in fractions with a specific composition in terms of sperm numbers and seminal plasma (SP), which is reflected in the varying sperm cryotolerance observed among different fractions. As boar sperm are particularly sensitive to oxidative stress, this study evaluated the role of SP antioxidants in the observed differences in sperm cryotolerance among ejaculate fractions. Ten ejaculates from five boars were manually collected in fractions: the first 10 mL of the sperm-rich fraction (SRF), the rest of the SRF and the post-SRF. Semen samples comprising the entire ejaculate (EE) were created by proportionally mixing the three fractions described above. Each of the 40 resulting semen samples was split into two aliquots: one was used for sperm cryopreservation following a standard protocol utilizing 0.5-mL straws, and the other was used to collect SP for antioxidant assessment. Frozen-thawed (FT) sperm from the SRF (the first 10 mL of the SRF and the rest of the SRF) and those from post-SRF were of the highest and worst quality, respectively, which was measured in terms of total and objective progressive motility and viability (P < 0.01). Viable FT sperm from the post-SRF generated more reactive oxygen species and experienced more lipid peroxidation than those from the SRF (both the first 10 mL and the rest of the SRF) (P < 0.01). The percentage of FT sperm exhibiting fragmented nuclear DNA did not differ among ejaculate fractions and the EE. Catalase, glutathione peroxidase and glutathione peroxidase 5 (GPx-5) were lowest in SP from the first 10 mL of the SRF (P < 0.001), whereas superoxide dismutase (SOD) and paraoxonase 1 (PON-1) were highest in SP of the SRF (both the first 10 mL and the rest of the SRF) (P < 0.01). Trolox-equivalent antioxidant capacity (TEAC) and the ferric-reducing ability of plasma (FRAP) were highest in SP from the first 10 mL of the SRF and lowest in the post-SRF (P < 0.001), whereas cupric-reducing antioxidant capacity was lowest (P < 0.05) in SP from the first 10 mL of the SRF. Regression analyses indicated that certain SP antioxidants had good predictive value for post-thaw recovery rates of total motility (R2 = 54.8%, P < 0.001; including SOD, TEAC and FRAP) and viability (R2 = 56.1%, P < 0.001; including SOD, PON-1, GPx-5 and TEAC). These results demonstrated that certain SP antioxidants are positively involved in boar sperm cryotolerance, minimizing the oxidative stress imposed by cryogenic handling.

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

Cryopreservation of boar semen is of growing interest for breeding companies. Overnight-shipping of pre-diluted ejaculates to specialized laboratories offers a practicable method, but requires fine-tuned protocols. In this study, the impact of holding post shipping at 17°C for 2 or 24h (n=10 samples) and of equilibration in lactose-egg yolk extender without glycerol at 5°C for 2, 4, 24 or 48h (n=11 samples) before freezing was investigated. Sperm-rich fractions of ejaculates from 21 mature Pietrain boars were collected at a single boar stud. After pre-dilution (1+1, v:v) with Beltsville thawing solution, samples were sent to the laboratory. Temperature profiles during transport and initial equilibration time were recorded. Semen quality post-thaw (PT) was evaluated using CASA and flow cytometry. Holding of 2h after shipping resulted in higher sperm motility (P=0.013) and beat cross frequency (BCF; P=0.047) compared to 24h. Differences between both groups vanished with prolonged incubation at 38°C PT. Equilibration at 5°C for 4h yielded the highest motility and BCF, whereas the equilibration for 48h impaired sperm motility. Membrane integrity, mitochondrial activity and DNA fragmentation index were not affected by any protocol modification. In conclusion, processing of pre-diluted boar semen shipped overnight within 2h after arrival at the laboratory is preferred to 24h of additional holding at 17°C. Extending the equilibration period in lactose-egg yolk extender without glycerol at 5°C from 2h to 4h before freezing is recommended.

Boar variability in sperm cryo-tolerance after cooling of semen in different long-term extenders at various temperatures

This study investigated individual boar variability in the quality of pre-freeze (PF) and post-thaw (PT) semen cooled in different long-term (LT) extenders and for different holding times (HT). Sperm rich fractions were diluted with Androhep® Plus (AHP), Androstar® Plus (ASP), Safecell® Plus (SCP) and TRIXcell® Plus (TCP) extenders, stored for 2h at 17°C (HT 1) and additionally for 24h at 10°C (HT 2) and the samples were subsequently evaluated and frozen. Besides the analysis of CASA sperm variables, mitochondrial membrane potential (MMP), plasma membrane integrity (PMI), normal apical ridge (NAR) acrosome integrity, and viability (YO-PRO-1−/PI−) of sperm were assessed in the PF and PT semen. Results indicated that boar, extender and HT group affected the sperm quality characteristics. There were great variations in PMOT and the sperm motion patterns of the PF semen among the boars. Differences in the HT groups of the PF semen, with respect to the sperm membrane integrity, were less marked among the boars. Consistent variations in TMOT and PMOT in the PT semen were observed among the boars, being greater in the HT 2 group. Most of the CASA-analyzed sperm motion patterns were greater in the HT 2 group of the PT semen. Furthermore, sperm MMP, PMI and viability were greater in the HT 2 group of the PT semen in most of the boars, while consistent differences were observed among the boars for sperm NAR acrosome integrity in either HT group. The significant effect of the cryopreservation process on the sperm membrane proteome was evident from the number of protein bands, detected in the electrophoretic profiles of sperm of the HT 1 and HT 2 groups. The electrophoretic profiles of the PF and PT semen among boars with poor and good semen freezability, however, differed with respect to the abundance and types of sperm membrane-associated proteins. The overall results of this study provided evidence that there are differences among boars in response to the different cooling regimens, and that cooling of extended semen for a 24-h period at 10°C modulated the functions of sperm in an extender-dependent manner, rendering the cells less susceptible to cryo-induced damage. It is suggested that the findings of this study have the potential to improve the technology of boar semen cryopreservation.

Serum testosterone, sperm quality, cytological, physicochemical and biochemical characteristics of the prostatic fraction of dogs with prostatomegaly

Prostatomegaly is a common finding in older non-neutered dogs. This study compared the serum testosterone, sperm quality and characteristics of the prostatic fraction between healthy dogs and dogs with prostatomegaly. Blood samples of ten dogs (five dogs from each group) were taken for serum testosterone measurement. Sperm motility, vigour, concentration, viability, membrane functionality and morphology were analysed in sperm-rich fraction. Osmolality, pH, cell types, and albumin, haemoglobin, acid phosphatase, alkaline phosphatase, glucose, triglycerides, cholesterol, calcium, phosphorus, magnesium and chloride were analysed in prostatic fraction. Dogs with prostatomegaly have the lowest sperm motility, vigour, concentration and functional membrane. Dogs with prostatomegaly have the highest glucose, triglycerides and cholesterol. Glucose was the only constituent positively correlated with serum testosterone and prostate volume. It can be concluded that dogs with prostatomegaly have poorer sperm quality, and glucose, triglycerides and cholesterol in prostatic fraction can be used as prostatomegaly biomarkers.

Effect of two freezing extenders on characteristic of fresh and frozen-thawed semen in endangered Old Kladruber stallions - A pilot study

The aim of the study was to evaluate the effect of two different extenders on sperm characteristic before equilibration and post-thaw in the endangered Old Kladruber stallions. Also, the response of individual stallions to the extenders used was tested. Semen was collected from six stallions every other day within one week. After centrifugation of the collected sperm-rich fraction, the supernatant was removed and sperm pellets were divided to two aliquots; these were diluted either with Gent (Minitube, Germany) or privately manufactured lactose-EDTA-egg yolk extender (Lact). Three cryopreserved insemination doses (IDs) from each extender (Gent and Lact) were prepared for each stallion from one collection (108 samples from six stallions in total). As a parameter of quality, the motility (computer assisted sperm analysis), viability (fluorescence staining), and morphology (eosin/nigrosine staining) were evaluated after dilution with freezing extenders (fresh) and after thawing (frozen-thawed). The different effects of chosen extenders on the quality of fresh semen were only manifested in higher kinematic parameters of sperm when the Lact extender was used. However, in frozen-thawed samples, the Gent extender yielded significantly better results in all of the evaluated parameters. The representation of sperm subpopulation was significantly influenced by extender in fresh as well as frozen-thawed samples; moreover, we found a significant effect of freezing on the distribution of these subpopulations. The response of individual stallions to chosen extenders was evident in the different quality of fresh as well as frozen-thawed IDs; Gent extender yielded better frozen-thawed IDs. Based on our results, among others describing quality parameters of ejaculate in endangered Old Kladruber stallions, we can recommend using Gent extender for the production of frozen-thawed IDs.

Freezing dog semen using −80 °C ultra-freezer: Sperm function and in vivo fertility

Long term storage of canine frozen semen is conventionally performed in liquid nitrogen (LN2). However, previous works in freezing canine semen using a −80 °C ultra-freezer (−80°C-UF) showed no differences on sperm quality after thawing. The main objective of this study was to compare the effects of the freezing techniques using LN2 or -80°C-UF on sperm function and in vivo fertility of frozen–thawed dog semen. The sperm-rich fraction of the ejaculate was collected separately from five Chihuahua breed, and each one divided into two aliquots, and frozen and stored in LN2 or -80°C-UF. Sperm function was analyzed for motility and viability, acrosome integrity, mitochondrial function and phosphatidylserine translocation by flow cytometry before and after cryopreservation. A total of 10 bitches were intravaginal inseminated (IVAI; LN2 frozen-thawed semen = 5 and −80°C-UF frozen–thawed semen = 5). Pregnancy status was confirmed 30 d after IVAI by transabdominal ultrasonography and live born puppies at term were recorded. Sperm function parameters were affected for both freezing protocols. Differences (P < 0.05) were found between freezing and storage methods in most of the parameters of sperm function analyzed, except in the phosphatidylserine translocation. The percentages of pregnancies were not different between the two freezing and storage protocols used. Semen freezing and storage using −80 °C UF is an effective technique for long-term preservation of canine spermatozoa.

Profile and reproductive roles of seminal plasma melatonin of boar ejaculates used in artificial insemination programs.

Melatonin (MLT) is present in seminal plasma (SP) of mammalian species, including pigs, and it is credited with antioxidant properties. This study aims to identify the sources of variation and the role of boar SP MLT on sperm quality and functionality and in vivo fertilizing ability of liquid-stored semen doses used in AI programs. The SP MLT was measured using an ELISA kit in a total of 219 ejaculates collected from 76 boars, and reproductive records of 5,318 AI sows were recorded. Sperm quality was assessed according to motility (computer-aided sperm analysis) and viability (cytometry evaluation). Sperm functionality was assessed according to the cytometric determination of intracellular HO generation, total and mitochondrial O production, and lipid peroxidation in liquid AI semen samples stored at 17°C over 144 h. The concentration of SP MLT differed among seasons ( < 0.01) and day length periods ( < 0.001) of the year, demonstrating that the ejaculates collected during the increasing day length period (9.80 ± 1.38 pg/mL, range: 2.75-21.94) had lower SP MLT concentrations than those collected during the decreasing day length period (16.32 ± 1.67 pg/mL, range: 5.02-35.61). The SP MLT also differed ( < 0.001) among boars, among ejaculates within boar, and among portions within the ejaculate, demonstrating that SP from the first 10 mL of sperm-rich ejaculate fraction (SRF) exhibited lower MLT concentrations than post-SRF. The SP MLT was negatively related ( < 0.001) to mitochondrial O production in viable sperm. The SP MLT did not differ among AI boars ( = 14) hierarchically grouped according to high and low fertility outcomes. In conclusion, SP MLT concentration in AI boars varies depending on the season of ejaculate collection and differs among boars, ejaculates within boar, and portions within ejaculate. The SP MLT may act at the mitochondrial level of sperm by reducing the generation of O. However, this antioxidant role of SP MLT was not reflected in sperm quality or in vivo fertility outcomes of AI semen doses.

Effect of boar ejaculate fraction, extender type and time of storage on quality of spermatozoa.

The aim of this study was to investigate the effect the sperm-rich fraction (F1) and the post-F1 fraction (F2) on the quality of boar spermatozoa stored in a liquid state. Ejaculates were collected from three Polish Landrace boars. Each ejaculate fraction was diluted with BTS short-term extender and Safe-Cell Plus (SCP) long-term extender and stored for seven days (D1-D7) at 17°C. Analyses included sperm motility parameters, normal apical ridge (NAR) acrosomes and plasma membrane integrity (PMI). Prior to the dilution of fractions, marked changes (p<0.05) were noted between F1 and F2 in progressive motility (PMOT), velocity average pathway (VAP) and velocity straight line (VCL). After the ejaculate was diluted, the type of fraction and type of extender significantly affected (p<0.05) PMOT, being markedly higher (p<0.05) for F1 extended in BTS. No marked changes (p<0.05) were observed between F1 and F2 extended in SCP for any of the analyzed sperm quality parameters during seven days of storage. Significantly higher (p<0.05) values of sperm quality parameters were noted in F1 compared with F2 for BTS on D7 of storage. The results of the four-way ANOVA analysis indicate that boar, fraction of ejaculate, extender type and day of storage had significant effects on the quality of boar stored spermatozoa. The F1 was characterised by higher quality of spermatozoa during storage in comparison with F2 in the short-term extender. Using the long-term extender containing the proteins allowed for a better application of F2, which could be important for the pig industry.

Proteomic characterization of canine seminal plasma

The present study was conducted to identify the major proteome of the sperm-rich fraction and prostatic fraction of canine seminal plasma. Three semen samples from four healthy dogs were obtained by digital manipulation. The pre-sperm fraction, sperm-rich fraction and prostatic fraction were separated from each ejaculate. Immediately after sperm analysis, a protease inhibitor was added to the sperm-rich fraction and prostatic fraction, and the fractions were separately centrifuged and frozen at −80 °C. The samples were thawed, re-centrifuged, and the total protein concentration was determined. Samples were subjected to 1D SDS-PAGE and Coomassie-blue stained gels, were analyzed by Quantity One 1D Analysis Software. Bands detected in the gels were excised and proteins subjected to digestion with trypsin. Proteins were identified by nano-HPLC-MS and tools of bioinformatics. Tandem mass spectrometry allowed the detection of 268 proteins in the gels of sperm-rich fraction and prostatic fraction of canine ejaculate. A total of 251 proteins were common to the sperm-rich and prostatic fractions, while 17 proteins were present in the sperm-rich fraction and absent in the prostatic fraction. The intensity of the bands detected in range 1 and 2 represented 46.5% of all of the band intensities detected in the 1D gels for proteins of the sperm-rich fraction and 53.0% of all bands in the prostatic fraction. Arginine esterase and lactotransferrin precursor were the protein with the highest intensity observed in the both fractions. Among the proteins present only in the sperm-rich fraction, the proteins UPF0764 protein C16orf89 homolog and epididymal-specific lipocalin-9 were the most abundant. In conclusion, canine sperm-rich fraction and prostatic fraction express a very diverse set of proteins, with unique biochemical properties and functions. Moreover, although most proteins are common to both sperm-rich fraction and prostatic fraction, there are some exclusive proteins in sperm-rich fraction.

Physical characteristics and fertility of fractionated donkey semen cooled at 5°C

ABSTRACT The aim of this study was to evaluate the effect of two different extenders (Skimmed Milk Glucose - SMG or Lactose - Egg Yolk - LEY) on physical characteristics and fertility of fractionated donkey semen cooled at 5°C. For this, four Pêga donkeys were used as semen donors. The sperm rich fraction of the ejaculate was diluted preparing insemination doses containing 400 x 106 motile spermatozoa in a volume of 22 mL, cooled to 5°C and stored up to 48 hours in a container proposed by Palhares (1997). Sperm motility and vigor were assessed in fresh semen, after first semen dilution, before insemination, at 24 and 48 hours after storage. For the fertility evaluation, 44 mares were inseminated with semen stored for a period between 12 and 24 hours. The mares were inseminated on fixed days (Mondays, Wednesdays, and Fridays) after the detection of a follicle greater than a 30mm diameter in one of the ovaries through ovulation. Pregnancy diagnosis was performed on day 12 post-ovulation, using transrectal ultrasonography. Semen diluted in SMG showed superior sperm motility than LEY, at the Pre-AI evaluation (P&lt;0.05). At 48 hours of storage, all donkeys had motility values between 45 and 53% for semen diluted in SMG, while only one donkey showed motility greater than 30% in the LEY treatment. The pregnancy rate/cycle for mares inseminated with semen diluted in SMG was superior than that obtained using LEY (56.52% vs 4.76%, respectively).