Semen Extender Research Articles

Effects of gelatin and oxytocin supplementation in a long-term semen extender on boar semen quality and fertility potential.

This study investigated the efficacy of different concentrations of gelatin supplementation in long-term semen extender on boar semen quality during storage for 10 days at 17°C. Additionally, oxytocin was added to stored semen to enhance fertility. In Experiment 1, boar semen was collected, diluted with gelatin at concentrations between 0% and 2.5% (w/v) and mixed with a semen extender. Then, it was kept in a refrigerator at 17°C and stored for 10 days. In Experiment 2, the sperm quality was examined after adding 0, 5, and 10 IU oxytocin per artificial insemination dose to the most effective semen extender from Experiment 1 and placing it in a refrigerator at 17°C for 10 days. In Experiment 3, the fertility potential in terms of non-return rate and litter size was determined using the most effective solid-stored semen supplemented with oxytocin. The results indicated that sperm quality decreased with increasing storage time (p<0.05). The sperm quality in terms of total motility, progressive motility, and viable sperm with intact acrosomes and high mitochondrial potential was the highest with 1.5% gelatin supplementation (p<0.001) on all days of storage. Treatment with oxytocin did not affect sperm quality (p>0.05). The non-return rate and litter size after insemination with semen supplemented with 1.5% gelatin and 10 IU of oxytocin after 8 to 10 days of storage were comparable to those of the control group (p>0.05). A semen extender as a solid medium supplemented with 1.5% gelatin successfully preserved boar semen for a long storage duration. Treatment with oxytocin did not affect sperm quality. In addition, the fertility capacity using 1.5% gelatin with 10 IU oxytocin and stored for 8 to 10 days was acceptable and comparable to that of short-term storage.

Ram semen quality after supplementation with gelatin, agar or alginate prior to cooling storage.

Adding gelling agents to convert the liquid state of the semen extender to a solid state allows for an increased sperm life span. Gelatin and alginate have been used to study the effects of gelling agents on sperm quality. However, there are other gelling agents that have not been studied, such as agar. In addition, studying different sources of gelling agents or the effect of mixing more than one gelling agent with semen extenders on sperm fertility has received little attention. Therefore, the objective of this study was to evaluate the effect of adding agar and a mixture of gelling agents from different sources to semen extender on ram sperm traits and fertility. The first trial evaluated the effect of the addition of 2.5-3 mg mL-1 of gelatin mixed with 0.5-20 mg mL-1 of agar or alginate to ram semen extender on sperm (motility, progressive motility, live/dead, membrane integrity) and semen (pH) characteristics. The response variables were evaluated 1, 72 and 144 h after storage at 4°C. In the second trial, two sources (feed grade and bacteriological) of gelatin and agar were evaluated on the response variables as in Trial 1. In trial 3, a total of 34 ewes were inseminated with doses supplemented (n = 17) with or without (n = 17) agar and gelatin. The pregnancy rate was diagnosed 40 days after insemination. In general, adding agar and gelatin improves (p < .05) sperm motility, membrane integrity and the ratio of live sperm after 144 h of storage compared to the Control group, regardless of the source (bacteriological or feed grade). However, the pregnancy rate in ewes was not influenced (p ≥ .05) by semen doses stored with agar and gelatin. In conclusion, the addition of agar and gelatin preserves ram sperm motility and membrane integrity after 144 of storage at 4°C without affecting the pregnancy rate in inseminated ewes.

Short-Term Storage of Rooster Ejaculates: Sperm Quality and Bacterial Profile Differences in Selected Commercial Extenders.

Bacterial contamination of semen has become an important contributor to the reduced shelf life of insemination doses in the poultry industry, which is why antibiotics (ATBs) are an important component of semen extenders. Due to a global rise in antimicrobial resistance, the aim of this study was to assess the efficiency of selected commercially available semen extenders to prevent possible bacterial contamination of rooster ejaculates. Two selected extenders free from or containing 31.2 µg/mL kanamycin (KAN) were used to process semen samples from 63 healthy Lohmann Brown roosters. Phosphate-buffered saline without ATBs was used as a control. The extended samples were stored at 4 °C for 24 h. Sperm motility, viability, mitochondrial activity, DNA integrity and the oxidative profile of each extended sample were assessed following 2 h and 24 h of storage. Furthermore, selective media were used to quantify the bacterial load and specific bacterial species were identified with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The results indicate that semen extenders enriched with KAN ensured a significantly higher preservation of sperm quality in comparison to their KAN-free counterparts. Bacterial load was significantly decreased in diluents supplemented with ATBs (p ≤ 0.001); however, KAN alone was not effective enough to eradicate all bacteria since several Escherichia coli, Enterococcus faecalis, Enterococcus faecium and Micrococcus luteus were retrieved from samples extended in KAN-supplemented commercial extenders. As such, we may suggest that more focus should be devoted to the selection of an optimal combination and dose of antibiotics for poultry extenders, which should be accompanied by a more frequent bacteriological screening of native as well as extended poultry semen.

Myo-inositol improves the viability of boar sperm during liquid storage.

Liquid preservation of boar semen is a highly preferred method for semen preservation in pig production. However, oxidative stress is the main challenge during the liquid preservation of boar semen in a time dependent manner. Therefore, supplementation of sperm with antioxidants during storage to protect them from oxidative stress has been the focus of recent research. Myo-inositol (Myo-Ins), the most active form of inositol, which belongs to the vitamin (Vit.) (B1 group has been shown to improve semen quality) (1). This study aimed to investigate whether Myo-Ins supplementation protects boar sperm in liquid preservation against oxidative stress and determine the appropriate concentration of Myo-Ins to be used in this regard. Boar sperm was diluted with a semen extender with different concentrations of Myo-Ins (2, 4, 6, and 8 mg/mL) depending on the previous studies (1, 24). Sperm motility and viability, plasma membrane and acrosome integrity, mitochondrial membrane potential (MMP), semen time survival, and gene expression were measured and analyzed on days 0, 1, 3, 5, and 7 for the different samples. Different concentrations of Myo-Ins exerted different protective effects on the boar sperm quality. The addition of 2 mg/mL Myo-Ins resulted in higher sperm motility and viability, plasma membrane and acrosome integrity, MMP, and effective survival time. Investigation of mRNA expression patterns via qRT-PCR suggested that the 2 mg/mL Myo-Ins sample had increased expression of antioxidative genes. The addition of Myo-Ins to semen extender improved the boar semen quality by decreasing the effects of oxidative stress during liquid preservation at 17°C. Additionally, 2 mg/mL is the optimum inclusion concentration of Myo-Ins for semen preservation.

Correlation between fatty acids levels in chicken, duck, goose, pigeon, quail and turkey egg yolks and post-thawed quality of ram semen.

The comparison between adding egg yolks (EY) of chicken, duck, goose, pigeon, Japanese quail or turkey to the Tris glycerol extender on the quality of ram semen before freezing and post-thawing was evaluated. The correlation between fatty acids levels in egg yolks of different avian species and the post-thawed quality of ram semen was studied. The pooled ejaculates collected from five rams were extended with Tris EY glycerol extenders containing the EY of chicken, duck, goose, pigeon, quail or turkey and cryopreserved at -196°C. The straws were evaluated before freezing and post-thawing for sperm motility using a sperm cell analyser, vitality using a FluoVit kit and abnormality using a SpermBlue stain besides plasma-membrane and DNA integrities using a hypo-osmotic swelling test and a Halomax kit, respectively. The moisture, ash, protein and fatty acid (FA) contents of EY of chicken, duck, goose, pigeon, quail and turkey were analysed using a gas chromatograph. The chicken and quail EY extenders significantly improved the total progressive motility (32.05 ± 1.41 and 31.68 ± 1.43, respectively), vitality, plasma membrane and DNA integrities and abnormalities of post-thawing ram semen in comparison with other EY extenders. Pigeon EY had the lowest saturated fatty acids (SFAs) in comparison with the other types of EYs. The chicken and turkey EYs had the lowest percentage of (monounsaturated fatty acids) MUFAs in comparison with the other types of EYs. The highest percentage of polyunsaturated fatty acids (PUFAs) was observed in the turkey, pigeon and chicken EYs which were considered double or triple their percentage in duck and goose EYs, respectively. Significant positive correlations existed between SFAs levels and total motility, vitality, plasma membrane functionality and DNA integrity (0.77, 0.80, 0.67, 0.52, respectively). Significant negative correlations existed between gondoic EY levels and total motility, vitality, plasma membrane functionality and DNA integrity. In conclusion, the EYs of duck, goose, pigeon or turkey cannot substitute the chicken EY in ram semen extenders as they gave lower post-thawing quality. The quail EY can be used as a good replacer for chicken EY in the extender used for cryopreservation of ram semen. The EY composition of FAs can significantly affect the quality of ram semen post-thawing.

Semen characteristics, nutrient utilization and immuno-endocrine status in response to dietary supplementation of a novel trace mineral mixture in crossbred bulls

BackgroundMicronutrients have a well-established role in the productive and reproductive health of animals, and there are many proven benefits of supplementing them in the diet of bulls or semen extender per se. Trace minerals have a key role in regulating male fertility and sperm function. MethodologyTwelve crossbred bulls were divided into two groups (n = 6): T1(control) and T2, (BullTraMin) and given diets with or without supranutritional supplementation of the novel trace mineral (BullTraMin), respectively. The experiment was conducted for 120 days and the effects on nutrient utilization, immuno-endocrine status, sexual behaviour and semen characteristics were investigated. Results and conclusionResults showed that body weight, dry matter intake, and nutrient digestibility were not affected by dietary supplementation with BullTraMin. Testosterone levels were increased (p < 0.05), while thyroxine hormone concentrations were unaffected (p < 0.05) in control (T1) and supplemented (T2) groups. Levels of trace minerals (Zn, Cu, and I) were improved in the blood and seminal plasma (p < 0.05) of the T2 group, while levels of Cr and Mn were increased only in the blood plasma. Sperm functions viz., viability, membrane integrity, acrosome integrity, motility, and mitochondrial membrane potential were improved (p < 0.05) due to BullTraMin supplementation. Furthermore, lipid peroxidation and membrane scrambling which impair the cryopreservilibity of spermatozoa, were reduced (p < 0.05) in T2 group. Blood antioxidant status (total antioxidant activity, superoxide dismutase and glutathione peroxidase levels) was improved (p < 0.05) in T2 group. Sexual behaviour was also improved (p < 0.05) in BullTraMin-supplemented group. Hence, it can be concluded that BullTraMin supplementation at a dose rate of 889 mg/kg diet (dry matter intake) is beneficial in improving hormonal status, antioxidant status, sexual behaviour and semen quality.

Melatonin supplementation to the freezing medium enhances post-thaw sperm quality and fertility of giant grouper (Epinephelus lanceolatus)

Giant grouper (Epinephelus lanceolatus) is not only favored in the international live reef-fish trade but also a promising species for mariculture in coastal communities throughout Asia. Sperm cryopreservation contributes to the extensive utilization of artificial insemination in grouper aquaculture industry. Inevitable damage during the process of freezing can negatively affect post-thaw sperm quality and fertility, thus hindering its routine application in practice. Melatonin is an antioxidant that can protect sperm from the adverse effects of cryopreservation in a variety of species. This study was aimed to assess the effect of melatonin addition on post-thaw sperm function of giant grouper. Semen samples were cryopreserved with freezing medium containing different final concentrations of melatonin (1, 2 and 4 μM) and the control group (without melatonin). After thawing, multiple sperm functional parameters were evaluated. Generally, a positive effect on cryopreservation was observed for all doses of melatonin. Melatonin treatment at 1 and 2 μM seemed to be efficient in preventing sperm cryodamage, which yielded significantly higher total motility, progressive motility, mitochondrial membrane potential (P < 0.05) and improved velocity (VCL, VAP, VSL) compared to controls. Lower apoptotic-like changes were detected at 2 μM treatment. Samples frozen with melatonin also contributed to greater sperm viability and lesser DNA damage. Although not statistically different from control, a reduction in intracellular ROS was found under the melatonin treatments. Moreover, melatonin supplementation improved the fertility of frozen-thawed sperm, which increased significantly the hatching rate under a limited sperm/egg ratio (10,000:1). Hence, our study suggests inclusion of melatonin to semen extender can improve the post-thaw quality of cryopreserved giant grouper spermatozoa, the recommended concentration of melatonin is 1–2 μM in the cryomedium.

Effect of In Vitro Addition of Melatonin and Glutathione on Seminal Parameters of Rams in Diluted Semen and after Thawing.

The present study aimed to evaluate the effects of melatonin (MLT), glutathione (GSH), and their combination on ram semen quality after thawing. During eight weekly sessions, semen from three Merino rams was pooled, diluted with an egg-yolk-based semen extender, and divided into four groups: control, 1 mM MLT, 5 mM GSH, and 1 mM MLT + 5 mM GSH. Diluted semen was evaluated before and after the freezing process. The supplementation of diluted semen with GSH at 5 mM had a deleterious effect on total motility progressive (TPM), curvilinear velocity (VCL), straight-line velocity (VSL), average-path velocity (VAP), linearity (LIN), and straightness (STR) and increased slow spermatozoa (%). MLT at 1 mM also had a negative effect on TPM, VSL, and STR in diluted semen. In thawed semen, 1 mM MLT increased the TPM compared with the control group. VSL was lower in the 5 mM GSH group than in the 1 mM MLT group. Additionally, the combination of both antioxidants attenuated the negative effect of 5 mM GSH on TPM, VSL, and BCF. These results indicate that 5 mM GSH impairs or does not improve sperm kinetic parameters in either diluted or thawed semen. They also suggest that MLT combined with GSH plays a protective role against these effects.

Improvement of post-thaw quality and fertilizing ability of bull spermatozoa using Rho kinase inhibitor in freezing extender.

In this study, it was hypothesized that the addition of an appropriate concentration of Y-27632 (a ROCK inhibitor) to the freezing extender prevents cryopreservation-induced apoptosis and improves embryonic development after in vitro fertilization (IVF). Semen samples were collected from five fertile Simmental bulls using an artificial vagina twice a week for 4 weeks. Selected samples were pooled and diluted with Tris-egg-yolk-glycerol (TEYG) extender containing different concentrations of Y-27632 (0, 10, 20, 30, and 40 μM) and then frozen in liquid nitrogen. After thawing, computer-assisted semen analysis (CASA), plasma membrane integrity, and acrosome intactness were evaluated in terms of morphological abnormalities, intracellular generation of reactive oxygen species (ROS), DNA fragmentation, phosphatidylserine (PS) externalization, and apoptotic-related gene expression. Finally, groups of frozen and thawed spermatozoa were used for bovine oocyte IVF. The results show that the semen extender at a concentration of 20 μM Y-27632 effectively improved total motility (TM), curvilinear velocity (VCL), as well as the plasma membrane and acrosome integrity compared to the control group (p < 0.05). Intracellular ROS levels were significantly (p < 0.05) lower in samples treated with 30 μM Y-27632 compared to the control specimen. Furthermore, supplementation of the semen extender with 20 μM Y-27632 resulted in more viable spermatozoa compared with the control group (p < 0.05). According to qRT-PCR results, the expression levels of BAX and CASPASE-9 genes in samples treated with 30 μM Y-27632 were significantly downregulated, while the expression of BCL2 was increased compared to the control (p < 0.05). The results of IVF demonstrated that the treatment of frozen-thawed spermatozoa with 20 μM Y-27632 increased blastocyst rates compared to the control group (p < 0.05). In conclusion, the addition of 20 μM Y-27632 into the freezing extender can improve the functionality and the fertilizing capacity of frozen spermatozoa due to its antioxidative and anti-apoptotic properties.

Assessment of the stallion sperm acrosome in two different extenders: Spermac stain in comparison with PNA/PSA/PI triple-staining.

Since the stallion acrosome is very small compared to other species and cannot be properly assessed without additional staining, several labelling techniques were developed to facilitate its assessment. The aim of this study was to compare the Spermac stain (Minitüb GmbH) and a PNA/PSA/PI triple-staining detected by flow cytometry with regard to method agreement for detecting non-intact acrosomes within two different extenders. For this purpose, eighteen stallion ejaculates were split in half and diluted with the semen extenders EquiPlus or Gent (Minitüb GmbH) to a final concentration of 50 × 106 sperm/mL, respectively. Subsequently, 126 semen samples were stained with both methods between 4 and 240 h (mean: 63.8 ± 48.9 h) after semen collection. Calculated Intraclass correlation coefficients revealed excellent correlations between both methods for EquiPlus (r = .77, p < .001) and fair correlations for Gent (r = .49, p < .001). Interestingly, flow cytometry detected more non-intact acrosomes in EquiPlus than in Gent (p < .001), whereas the Spermac stain showed no differences (p = .902) between extenders. The poorer method agreement in Gent could be caused by egg yolk artefacts, which made interpretation difficult, so flow cytometry might be preferred. The differences in detected non-intact acrosomes between extenders highlighted the importance of establishing adapted laboratory protocols for different extender types in order to generate comparable results.

Trends in rabbit insemination extenders for fresh and frozen semen. A review

Artificial insemination (AI) has become a popular technique in rabbit farms worldwide. This report discusses the progress made on semen extenders used in rabbit AI, setting out the latest innovations. Fresh and frozen semen have different requirements, so the extender composition will vary depending on the type of semen used. We discuss the endocrine supplementation of extenders for ovulation induction, the use of active molecules as an alternative to conventional antibiotics and the extenders developed for rabbit sperm cryopreservation.

Effects of propolis-loaded nanoliposomes fortification in extender on buffalo semen cryopreservation

Buffalo sperm is sensitive to cryoinjuries, thus improving sperm cryoresistance is a critical approach for wide spreading the assisted reproductive technologies in buffalo. The intention of this work was to assess the effect of propolis-loaded in nanoliposomes (PRNL) supplementation of semen extender on semen quality, antioxidant status and some apoptotic genes of cryopreserved buffalo semen. PRNL were prepared using cholesterol (Chol) as well as soybean lecithin and their physicochemical properties were characterized. Egyptian buffalo bulls (4–6 years) were involved, and the semen samples were collected using the artificial vagina method. Buffalo semen was pooled (n = 25 ejaculates) and cryopreserved in tris extender containing PRNL at 0 (PRNL0), 2 (PRNL2), 4 (PRNL4) and 6 µg/mL (PRNL6), respectively. The PRNL had a size of 113.13 nm and a negative zeta potential (− 56.83 mV). Sperm progressive motility, viability, membrane integrity, abnormalities, chromatin damage, redox status, apoptosis status, and apoptotic genes were investigated after post-thawed buffalo semen. Using 2 or 4 µg/mL PRNL significantly increased sperm progressive motility, viability, and membrane integrity, while sperm abnormalities and the percentage of chromatin damages were the lowest in PRNL2 group. Moreover, the PRNL2 group exhibited the best results for all antioxidative activities (TAC, SOD, GPx and CAT) with significantly higher levels than the other groups (P < 0.05). The levels of ROS and MDA were significantly lower in the PRLN2 compared with other groups. The sperm caspase 3 enzyme activities showed the lowest values in PRNL2 groups followed by PRNL4 and PRNL6 groups with significant differences compared with the control. Adding 2 µg/mL PRNL to freezing media significantly reduced apoptotic genes such as Bax and Caspase 3 in sperm, while significantly increase in Bcl2 expression compared with the control (P < 0.001). The expression of Bcl2, Caspase 3 and Bax genes in sperm were not affected by the 6 µg/mL PRNL addition (P > 0.05). The electron micrography descriptions exemplified that the fortification of 2 or 4 µg/mL PRNL maintained the acrosomal and plasma membrane integrities as well as sustained the ultrastructure integrity of the cryopreserved buffalo spermatozoa when compared with control group, whereas the 6 µg/mL of PRNL demonstrated highest injury to the acrosome and plasma membranes. Results show supplementation of the buffalo freezing extender with 2 or 4 µg/mL of PRNL enhanced post-thawed sperm quality via boosting the antioxidant indices, diminishing the oxidative stress and apoptosis as well as maintained the ultrastructure integrity of frozen-thawed buffalo sperm.

Motility of rabbit buck semen extended with quail-egg yolk and turkey egg yolk citrate as rabbit buck semen extenders at refrigerated temperature

Motility of rabbit buck semen extended with quail-egg yolk and turkey egg yolk citrate as rabbit buck semen extenders at refrigerated temperature

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.

Effects of Commercial and Homemade Extenders on Post-thaw Sperm Quality and Fertility of Semen from Ethiopian Indigenous Horro Chicken Breed

Cryopreservation of spermatozoa represents an important strategy for in vitro programs designed for the conservation of the genetic material of livestock populations. The objective of this study was to evaluate the effects of homemade tris-egg yolk-based and commercial poultry semen extenders on post-thaw sperm quality, fertility, and hatchability of semen from the Ethiopian Indigenous Horro chicken breed. A total of 30 roosters were used for semen collection, and 160 adult hens were inseminated artificially. The collected, qualified, and pooled semen samples were divided into three groups. Each semen sample was diluted with a homemade tris-egg yolk-based extender, Dimethyl-formamide commercial extender, and Commercial Beltsville Poultry Extender. Each extended semen was further divided into 20 sterile tubes as replicates. The extended semen samples were cryopreserved in liquid nitrogen using standard procedures. Changes in post-thaw spermatozoa mass and progressive motility, in vitro viability, morphological abnormality, fertility, and hatchability were evaluated. The post-thaw evaluation showed a decrease in the mass and progressive motility, morphologically normal spermatozoa, and an increase in dead spermatozoa and spermatozoa with bent necks, compared to fresh semen. There were significant differences in progressive sperm motility, motility, and in vitro viability between commercial and homemade cryoprotectants. However, no significant difference was observed in mass motility across the extenders. The commercial Dimethyl-formamide extender was found to be the most suitable regarding the proportion of morphologically normal sperm and in vitro viability rate of cryopreserved sperm samples. There were no significant differences across all treatments in terms of fertility and hatchability rate. However, there was a significant difference between the control treatment and commercial extenders in terms of fertility and hatchability. The findings indicated favorable outcomes for a tris-egg yolk-based extender that was prepared locally with regard to the cryopreservation of poultry semen. Additional investigations are recommended to enhance the fertility and hatchability of the semen.

Effect of Atomized Black Maca (Lepidium meyenii) Supplementation in the Cryopreservation of Alpaca (Vicugna pacos) Epididymal Spermatozoa.

Artificial insemination is an important assisted reproductive technology that has been applied in several mammalian species. However, successful cryopreservation of semen of South American camelids has been limited, hindering the commercial application of artificial insemination in alpaca species. In this scenario, the addition of antioxidants to semen extenders provides a strategy to improve the freezability of mammalian sperm. Bioactive metabolites from natural extracts of black maca have shown valuable antioxidant properties. Thus, the objective of this study was to evaluate the effect of the addition of atomized black maca in the freezing medium of epididymal spermatozoa of alpacas. Fifteen pairs of epididymis were collected from a local slaughterhouse. Each sample was divided into six groups: (1) fresh, (2) yolk medium (YM), (3) 10 mg/mL maca, (4) 20 mg/mL maca, (5) 30 mg/mL maca, and (6) resveratrol (as an antioxidant control). Sperm cryopreservation was performed through the slow freezing method. Markers associated with functionality, such as motility, viability, and plasma membrane integrity, as well as markers associated with oxidative damage, such as DNA integrity, total ROS production, and mitochondrial function, were analyzed. The results show that the supplementation with black maca (20 mg/mL) improved the sperm motility, viability, plasma membrane integrity, and mitochondrial function evaluated according to an index of formazan deposits. Similarly, the ROS production decreased with maca at 20 mg/mL, although the DNA integrity did not show any differences among the groups. These results suggest that maca at 20 mg/mL has cytoprotective effects during freezing/thawing of epididymal sperm of alpaca species. Further research will be focused on assessing the effects of maca supplementation on semen extenders by using biomolecular markers (proAKAP4) associated with fertility.

Epsilon poly-lysine in buffalo semen extender: A step towards reducing the development of antibiotic resistance.

The use of antibiotics in semen extenders can contribute to the development of antibiotic resistance. The objective of the study was to evaluate epsilon-polylysine (Ɛ-PL) as a substitute for antibiotics in the buffalo semen extender. For this, 20 semen ejaculates were collected from four Murrah buffalo bulls. Each ejaculate was divided into three equal aliquots and extended into an egg yolk-based semen extender containing either antibiotics (strepto-penicillin) or different concentrations of Ɛ-PL (0.64 and 1.28 g/L) to make the final concentration 80 million sperm/mL and cryopreserved as per the standard procedure. The antibiogram sensitivity test confirmed that Ɛ-PL is an effective antimicrobial against microbes present in buffalo semen ejaculates. Furthermore, the addition of Ɛ-PL in the semen extender significantly reduces the colony forming unit (CFU)/mL in cryopreserved semen equivalent to strepto-penicillin. The sperm motility and kinematic parameters assessed by a computer-assisted sperm analyser showed that Ɛ-PL did not inhibit either sperm motility not kinematic parameters of cryopreserved sperm. The flow-cytometric evaluation of frozen-thawed sperm revealed interesting results. The extender supplemented with Ɛ-PL protected sperm acrosome and mitochondrial membrane potential greater than the extender supplemented with strepto-penicillin. Further, Ɛ-PL reduced significantly the production of superoxide anions from mitochondria during the cryopreservation process. In this way, Ɛ-PL may be a suitable alternative to antibiotics in semen extenders. In conclusion, Ɛ-PL at a concentration of 0.64 g/L acts as an effective antimicrobial as well as antioxidant in semen extender for cryopreservation of buffalo sperm.

Double semen collection at a 1-h interval in dogs decreases the bacterial contamination of canine ejaculates

Semen extenders usually contain antibiotics with the aim to minimize bacterial growth, but the indiscriminate use of antibiotics increases the emergence of multidrug-resistant bacteria. A limiting factor of semen processing in dogs is the low total sperm count that limits the number of insemination doses that can be obtained from one ejaculate. Therefore, two ejaculates collected at a short interval can be combined to increase the number of AI doses. In this study, semen was collected from dogs either once or the same dogs (n = 28) were submitted to dual semen collection 1 h apart. All ejaculates were submitted to bacteriological analysis. We hypothesized that bacterial contamination of semen is low but that a dual semen collection might increase contamination. A sample for bacteriological examination was taken from raw semen immediately after semen collection. Bacteria including mycoplasmas were isolated using conventional cultivation procedures and isolates were identified to the species level by matrix-assisted laser desorption ionization – time of flight (MALDI-ToF) mass spectrometry. In total, 22 bacterial species were identified in the 84 ejaculates with Mycoplasma cynos, Streptococcus canis and Canicola haemoglobinophilus being most frequent. Bacterial growth was sporadic in 16 and absent in 10 ejaculates. The overall bacterial growth was lower in the second than in the first ejaculate of dual semen collections (p < 0.05). The percentage of motile and membrane-intact spermatozoa in frozen-thawed ejaculates was not associated with the degree of bacterial contamination of raw semen. In conclusion, there was only limited microbial contamination in dog semen and the microorganisms isolated are considered part of the normal genital bacterial flora. Repeated semen collection reduced bacterial contamination in the second in comparison to the first ejaculate. The use of antibiotics in canine semen should be questioned.

Effects of different semen extenders on sperm motility and pregnancy rates in donkeys

Semen quality is highly associated with the success of artificial insemination (AI). The characteristics of semen preserved at room temperature will be influenced by various factors. The semen extender provides sufficient energy for sperm, maintains pH and osmotic pressure stability, inhibits the reproduction of microorganisms and thus prolongs the survival time of sperm. We investigated effects of extender type on semen motility and pregnancy rates in donkeys. Five Yangyuan jacks and 1000 Yangyuan jennies were included in the study. Semen was collected by artificial vagina and filtered to remove impurities. The sperm motility (i.e., total and progressive) and concentration were determined by computer-assisted sperm analysis. Biggers, Whitter and Whittingham (BWW), Brackett and Oliphant medium (BO), Human Tubal Fluid (HTF) and Beltsville Thawing Solution (BTS), were semen extenders from other animal species, CAU-A and CAU-B were diluents independently developed by our laboratory. Firstly, the semen was diluted with CAU-B in a ratio of 1:2 (v/v, sperm: CAU-B). After mixing, the semen was centrifuged at 1500rpm for 10 min. The precipitation was retained and added to each tube. Then, the prewarmed extenders were slowly added along the tube wall and mixed by gentle shaking. After cooling to 17°C, diluted semen was placed in an incubator, mixed every 8h, and the sperm motility was analyzed. Meanwhile, we used transrectal palpation andB-mode ultrasound to determine ovarian activity of jennies. Upon detection of a 30-35 mm follicle, jennies were treated with the GnRH analogue deslorelin (GnRH-D) and inseminated with fresh semen diluted appropriately. The pregnancy rate of jennies was evaluated 20 days after ovulation. Statistical analysis was performed by ANOVA. Values are given as mean ± standard error. When diluted semen was stored at 17°C, the motility decreased with the increase of storage time extender. After 16h, sperm in BWW, BTS, BO and HTF extender were all dead. After 48h storage, the motility of sperm in CAU-A (mean 36.14 ± 6.12%) was lower (p < 0.05) than that in CAU-B (53.87 ± 16.25%). Pregnancy rates after insemination with semen diluted in CAU-A, CAU-B, BWW and BO were 30.53%, 34.77%, 29.37% and 30.96%, respectively, and there was no significant difference among groups (p>0.05). However, the pregnancy rate of semen diluted by CAU-B, which contains raffinose and fructose was slightly higher than that of the other groups. In conclusion, the results show that extension of donkey semen in CAU-B extender resulted in a slightly although not significantly improved pregnancy rate. This suggests that the semen extender is important for both, sperm viability and fertilization. This finding may be of significance for breeding and reproduction of donkeys and other equid species.

Evaluation of simple strategies to reduce the use of antibiotics in equine fresh sperm

According to the World Health Organization in 2021, the development of antimicrobial resistances is considered one of the top ten global public health threats. Microbial load negatively affects sperm quality and preservation (Ortega-Ferrusola and Peña, Reproduction in Domestic Animals. 2009; 44(3):518-522), which implies the use of antibiotics in semen extenders. The objective of this study was to study the effect of three semen processing techniques on quality and microbial load of fresh equine sperm to evaluate the possibility of reducing or eliminating the prophylactic use of antibiotics in the future. Ten adult stallions were included (one ejaculate per male). To avoid environmental contamination during sperm collection and processing, a protocol that maximizes hygiene measures was implemented. Aliquots of each ejaculate were processed following 4 protocols: Simple Centrifugation (450g x 7 min) in extender with antibiotics (SC+, as control group), Simple Centrifugation in extender without antibiotics (SC-), Single-Layer Colloidal Centrifugation (300g x 20 min) in extender without antibiotic (CC-) and Filtration (SpermFilter®) in extender without antibiotics (F-). Sperm motility (total and progressive motility, TM and PM, respectively, %), viability (Alives, %), and microbial load (on three different culture media (Columbia 5% Sheep Blood Agar, COS, general purpose medium for non-fastidious and fastidiousmicroorganisms, Sabouraud Dextrose Agar, SDA, for yeasts and molds, and Schaedler vitamin K1 5% Sheep Blood Agar, SCH, for fastidious anaerobic bacteria) were evaluated after processing. Statistical analysis was performed by Kruskal-Wallis, ANOVA and post hoc tests (p<0.05). Results are expressed as mean values ± SD. No significant differences were found between the control (SC+) and other groups (SC-, CC- and F-), neither in sperm characteristics (61,32a ± 20,84, 59,13a ± 21,84, 63,19a ± 18,87 and 52,54a ± 21,08 for PM and 84,60a ± 9,21, 85,05a ± 9,14, 84,90a ± 13,5 and 77,35a ± 13,27 for ALIVES respectively) nor in microbial load (216,50a ± 504,41, 348,30a ± 792,99, 731,30a ± 2180,11 and 51,70a ± 64,64 for COS; 1,00a ± 3,16, 11,00a ± 31,43, 4,00a ± 6,99 and 1,00a ± 3,16 for SDA and 25,00a ± 57,59, 41,00a ± 60,45, 10,00a ± 18,26 and 26,00a ± 42,48 for SCH respectively). The present processing methods, in combination with management measures that prevent environmental contamination, maintained sperm quality and microbial load of equine fresh semen to the same extent as traditional sperm processing protocols with antibiotics. Acknowledgements: Authors thank P. Cuesta for statistical assistance. This research was funded by Madrid Institute for Rural, Agricultural and Food Research and Development (IMIDRA).