Search

Simple Summary: Artificial insemination (AI) is a reproductive technique routinely used in livestock to deliver sperm directly to the cervix or uterus to achieve pregnancy. In swine, AI is performed with chill-stored sperm displaying minimum criteria evaluated using traditional procedures; however, variegated fertility appeals for additional tools for sperm evaluation. To enhance sperm evaluation, we assess the potential of near-infrared (NIR) and Raman spectroscopy in addition to traditional techniques to monitor boar sperm quality during 10-day chill-storage. Sperm samples showed differential responses to storage, with sperm quality nearly maintained in some samples, whereas it sharply deteriorated in others. Better than NIR, Raman spectral profiles obtained from freshly extended samples efficiently predict sperm survival to storage, making the technique a promising tool for in-depth assessment of sperm samples. Artificial insemination (AI) plays a critical role in livestock reproduction, with semen quality being essential. In swine, AI primarily uses cool-stored semen adhering to industry standards assessed through routine analysis, yet fertility inconsistencies highlight the need for enhanced semen evaluation. Over 10-day storage at 17 °C, boar semen samples were analyzed for motility, morphology, sperm membrane integrity, apoptosis, and oxidative stress indicators. Additionally, machine learning tools were employed to explore the potential of Raman and near-infrared (NIR) spectroscopy in enhancing semen sample evaluation. Sperm motility and morphology gradually decreased during storage, with distinct groups categorized as "Good" or "Poor" survival semen according to motility on Day 7 of storage. Initially similar on Day 0 of semen collection, "Poor" samples revealed significantly lower total motility (21.69 ± 4.64% vs. 80.19 ± 1.42%), progressive motility (4.74 ± 1.71% vs. 39.73 ± 2.57%), and normal morphology (66.43 ± 2.60% vs. 87.91 ± 1.92%) than their "Good" counterparts by Day 7, using a computer-assisted sperm analyzer. Furthermore, "Poor" samples had higher levels of apoptotic cells, membrane damage, and intracellular reactive oxygen species on Day 0. Conversely, "Good" samples maintained higher total antioxidant capacity. Raman spectroscopy outperformed NIR, providing distinctive spectral profiles aligned with semen biochemical changes and enabling the prediction of semen survival during storage. Overall, the spectral profiles coupled with machine learning tools might assist in enhancing semen evaluation and prognosis. [ABSTRACT FROM AUTHOR]

The longevity of sperm in teleost such as zebrafish and medaka is short when isolated even in saline-balanced solution at a physiological temperature. In contrast, some internal fertilizers exhibit the long-term storage of sperm, >10 months, in the female reproductive tract. This evidence implies that sperm in teleost possesses the ability to survive for a long time under suitable conditions; however, these conditions are not well understood. In this study, we show that the sperm of zebrafish can survive and maintain fertility in L-15-based storage medium supplemented with bovine serum albumin, fetal bovine serum, glucose, and lactic acid for 28 days at room temperature. The fertilized embryos developed to normal fertile adults. This storage medium was effective in medaka sperm stored for 7 days at room temperature. These results suggest that sperm from external fertilizer zebrafish and medaka has the ability to survive for at least 4 and 1 week, respectively, in the body fluid-like medium at a physiological temperature. This sperm storage method allows researchers to ship sperm by low-cost methods and to investigate key factors for motility and fertile ability in those sperm.

Phospholipase A (PLA) in goat semen aggregates with egg yolk in semen diluent, leading to sperm death. The aim of this study is to address the issue of sperm death caused by the interaction between PLA and egg yolk, and to explore the protective effect and metabolic regulation mechanism of the combination of dexamethasone (DXMS) and azithromycin (AZM) on goat sperm under low temperature conditions. At a low temperature of 4 °C, different concentrations of DXMS were added to semen diluents containing 30 μg/mL AZM to detect the quality of goat sperm. The optimal concentration of DXMS was determined to be 20 μg/mL. On the 5th day of storage, antioxidant capacity, total cholesterol (TC) levels, energy metabolism, and metabolomics analysis were performed on the sperm of the 20 μg/mL DXMS group. The results showed that there was no aggregation caused by the interaction between PLA and egg yolk in the group containing 30 μg/mL AZM at 4 °C. 20 μg/mL DXMS significantly improved sperm motility, plasma membrane integrity, acrosome integrity, glutathione peroxidase (GPX) (P < 0.05), catalase (CAT) (P < 0.01), and superoxide dismutase (SOD) activity (P < 0.01). The content of reactive oxygen species (ROS) and Fe2+ significantly decreased (P < 0.01), while the content of ATP (P < 0.01) and TC (P < 0.05) significantly increased. Through metabolomics analysis, a total of 56 differential metabolites (P < 0.05) were screened, including 5a, 6-Anhydrotetracycline, Betamethasone, and 11-Dehydrocorticosterone, mainly enriched in 8 metabolic pathways (P < 0.05), including steroid hormone biosynthesis, glycerophospholipid metabolism, and choline metabolism in cancer. Among them, 5 metabolic pathways are related to lipid metabolism. The results indicate that AZM effectively inhibits the aggregation of PLA and yolk, and the combination of AZM and DXMS enhances the preservation quality of goat sperm during low-temperature preservation by regulating lipid metabolism. [ABSTRACT FROM AUTHOR]

Abstract This study aimed to investigate if washing ram sperm from seminal plasma (SP) could be an effective tool to extend sperm lifespan in medium-term preservation in liquid form to optimize ovine artificial insemination protocols. To this end, in Experiment 1 SP was added to a sperm model without previous contact with this substance (ram epididymal sperm) at the beginning or the end of a 48-hour preservation protocol at 5 °C (n = 13). Sperm motility and kinetic parameters and sperm functionality in terms of sperm viability, apoptosis, mitochondrial activity and reacted acrosomes were assessed after 6 h of storage at 15 °C (standard liquid preservation method) and 24 and 48 h at 5 °C. Extended sperm showed better results after 48 h when stored in the absence than in the presence of SP in most sperm quality parameters. Moreover, the final SP supplementation of this experimental group resulted in the highest sperm motility and kinetic parameters, viability and mitochondrial activity. These results suggested that initial SP deprivation could be beneficial in a medium-term ram sperm preservation protocol in liquid form, as well as a final supplementation. Therefore, we conducted Experiment 2 to evaluate the effect of SP removal from freshly ejaculated ram semen under the same storage conditions as in Experiment 1 (n = 12). Surprisingly, SP withdrawal impaired sperm functionality, leading to increased apoptosis and decreased mitochondrial activity after 24 and 48 h at 5 °C. Conversely, SP supplementation at the end of the preservation protocol of the ejaculate processed as usual had a positive effect on sperm quality and fertility. To summarize, SP absence was beneficial for a medium-term preservation protocol (up to 48 h at 5 °C) of ram epididymal sperm, but the same preservation protocol for ram ejaculated sperm revealed a possible failure of the SP removal method in avoiding the sperm-SP interaction effect. Meanwhile, SP supplementation of ram semen at the end of the preservation protocol increased in vitro sperm quality and fertility after artificial insemination.

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. [ABSTRACT FROM AUTHOR]

This study aimed to investigate if washing ram sperm from seminal plasma (SP) could be an effective tool to extend sperm lifespan in medium-term preservation in liquid form to optimize ovine artificial insemination protocols. To this end, in Experiment 1 SP was added to a sperm model without previous contact with this substance (ram epididymal sperm) at the beginning or the end of a 48-hour preservation protocol at 5 °C (n = 13). Sperm motility and kinetic parameters and sperm functionality in terms of sperm viability, apoptosis, mitochondrial activity and reacted acrosomes were assessed after 6 h of storage at 15 °C (standard liquid preservation method) and 24 and 48 h at 5 °C. Extended sperm showed better results after 48 h when stored in the absence than in the presence of SP in most sperm quality parameters. Moreover, the final SP supplementation of this experimental group resulted in the highest sperm motility and kinetic parameters, viability and mitochondrial activity. These results suggested that initial SP deprivation could be beneficial in a medium-term ram sperm preservation protocol in liquid form, as well as a final supplementation. Therefore, we conducted Experiment 2 to evaluate the effect of SP removal from freshly ejaculated ram semen under the same storage conditions as in Experiment 1 (n = 12). Surprisingly, SP withdrawal impaired sperm functionality, leading to increased apoptosis and decreased mitochondrial activity after 24 and 48 h at 5 °C. Conversely, SP supplementation at the end of the preservation protocol of the ejaculate processed as usual had a positive effect on sperm quality and fertility. To summarize, SP absence was beneficial for a medium-term preservation protocol (up to 48 h at 5 °C) of ram epididymal sperm, but the same preservation protocol for ram ejaculated sperm revealed a possible failure of the SP removal method in avoiding the sperm-SP interaction effect. Meanwhile, SP supplementation of ram semen at the end of the preservation protocol increased in vitro sperm quality and fertility after artificial insemination. [ABSTRACT FROM AUTHOR]

During the freezing process of semen, due to the generating of significant amounts of free radicals, the quality of sperm changes. Epigallocatechin-3-gallate (EGCG) is a green tea catechin, which in this study was applied to investigate its effect on the quality of bulls' sperm. We collected semen samples with an artificial vagina from 12 Simmental bulls to evaluate the effect of EGCG (10.00 and 20.00 µmol) in two cryopreserving methods on the quality parameters of semen. We designed six groups including two control groups (method one and two) and four treatments (EGCG 10.00 µmol + method one; EGCG 20.00 µmol + method one; EGCG 10.00 µmol + method two; EGCG 20.00 µmol + method two). The 20.00 µmol EGCG and a method two significantly affected the amending oxidative conditions as well as an increase in total antioxidant capacity and a decrease in malondialdehyde. The effect of EGCG in both concentrations was more on method two. The desired impact on sperm motility, viability, inhibition of lipid peroxidation and sperm DNA damage was observed in EGCG groups compared to control groups. Among the two methods, the method two had fewer adverse effects on the plasma membrane, motility parameters, viability and DNA of sperm. The EGCG in the semen extender yielded a favorable impact on thawed sperm. This effect was prompted in combination with the method two. [ABSTRACT FROM AUTHOR]

The samples of small numbers of human sperm are not only difficult to obtain (testicular/ epididymal puncture surgery or microsurgical sperm extraction), but also few in number and poor in quality. Both the sperm recovery rate and the retrieval rate are low using traditional freezing methods. The existing technology of sperm cryopreservation has been improved in many aspects in order to enhance the cryopreservation effect of small numbers of sperm. The newly developed freezing vectors have made great progress in improving the recovery rate of motile sperm, the recovery rate of sperm and the convenience of operation. The cofactors of cryophosphate, such as antifreeze proteins, lecithin, nanoparticles and antioxidants, improved the cryophosphate effect to some extent. Sperm vitrification cryopreservation technology has gradually shown its development potential. This paper reviews the research progress of cryopreservation of small numbers of sperm, focusing on the optimization of cryopreservation vectors, cryopreservation protectant and cryopreservation program. [ABSTRACT FROM AUTHOR]

Simple Summary: This study focused on the importance of accurately assessing ram sperm quality for optimizing assisted reproductive technologies in sheep. Semen preservation can lead to sperm damage due to various stressors, including osmotic, biochemical, and thermal factors. To address this issue, the research aimed to determine the best time to evaluate ram sperm quality and identify the factor causing changes in sperm quality during liquid storage. In Experiment 1, ejaculated sperm were assessed for motility and functionality at different preservation times: 0, 3, 6, and 24 h. Both motility and functionality improved after 6 h. Experiment 2 delved into the responsible factor by using epididymal sperm. Results revealed that extender addition to the sperm caused altered motility at 0 and 24 h, and reduced functionality at 0 h. This suggests that the extender initially alters ram sperm, leading to sublethal damage that becomes reversible after 3 to 6 h of semen preservation. Thus, ram sperm require an adaptation period of 3 to 6 h to the extender before a precise quality assessment. This finding has practical implications for reproduction centers, allowing better workflow organization and optimal expression of ram sperm attributes at the time of cervical artificial insemination. Accurate assessment of ram sperm quality is crucial to optimizing assisted reproductive technologies in sheep. However, semen preservation can induce sperm due to osmotic, biochemical, and thermal stress. Stabilizing sperm with a suitable cooling rate and adaptation period to the extender could mitigate these effects for a more reliable evaluation. This study aimed to determine: (1) the best time to assess ram sperm quality, and (2) the factor responsible for the altered state of ram sperm during the first hours of liquid storage. In Experiment 1, ejaculated sperm were diluted and assessed for sperm motility and functionality at four preservation times: 0, 3, 6, and 24 h as sperm damage control. Both sperm motility and functionality improved after 6 h. Experiment 2 investigated the factor responsible for sperm quality change by testing the interactions of seminal plasma and extender with sperm from epididymides independently and in combination. The evaluation of sperm was performed as in Experiment 1. Sperm in groups containing the extender showed altered motility at 0 and 24 h, and lower functionality at 0 h. Thus, we could assume that extender addition initially alters ram sperm, causing sublethal damage that is reversible after 3 to 6 h of semen preservation. In conclusion, ram sperm require an adaptation time of 3 to 6 h to the extender before an accurate quality assessment can be conducted. This has practical implications for reproduction centers, enabling better workflow organization and optimal expression of ram sperm attributes when cervical artificial insemination is routinely performed. [ABSTRACT FROM AUTHOR]

Purpose: We compared semen quality and sperm DNA fragmentation in cancer patients who underwent sperm banking and controls who underwent sperm cryopreservation for assisted reproductive technology (ART). Materials and Methods: A total of 132 men, 65 cancer patients and 67 controls, were prospectively enrolled and performed sperm cryopreservation for fertility preservation from May 2019 to February 2021. Sperm quality was determined by measuring semen volume, sperm concentration, sperm motility, and sperm DNA fragmentation index (DFI). Sperm quality and sperm DFI were compared in cancer patients and controls. Results: The major cancers of the 65 cancer patients were leukemia (26.2%), testicular cancer (23.1%), and lymphoma (20.0%). Sperm concentration, sperm total motility, and sperm progressive motility were significantly lower in cancer patients than in controls. Sperm DFI was significantly higher in cancer patients than in controls (24.32%±15.69% vs. 19.11%±11.63%; p=0.033). After excluding 8 cancer patients who received chemotherapy before sperm banking, sperm concentration, sperm total motility, and sperm progressive motility were significantly lower in cancer patients than in controls, but there was no significant difference in sperm DFI for cancer patients and controls (23.14%±12.79% vs. 19.11%±11.63%; p=0.069). Conclusions: Sperm quality was lower in cancer patients than in controls. There was no difference in the sperm DFI of cancer patients prior to chemotherapy and men presenting for sperm cryopreservation for ART. We recommend that all men who are planning cancer therapy should be offered sperm banking prior to gonadotoxic chemotherapy as a standard of fertility preservation.

Klinefelter syndrome (KS) is characterized by a masculine phenotype, supernumerary sex chromosomes (usually XXY), and spermatogonial stem cell (SSC) loss in their early life. Affecting 1 out of every 650 males born, KS is the most common genetic cause of male infertility, and new fertility preservation strategies are critically important for these patients. In this study, testes from 41, XXY prepubertal (3-day-old) mice were frozen-thawed. Isolated testicular cells were cultured and characterized by qPCR, digital PCR, and flow cytometry analyses. We demonstrated that SSCs survived and were able to be propagated with testicular somatic cells in culture for up to 120 days. DNA fluorescent in situ hybridization (FISH) showed the presence of XXY spermatogonia at the beginning of the culture and a variety of propagated XY, XX, and XXY spermatogonia at the end of the culture. These data provide the first evidence that an extra sex chromosome was lost during innate SSC culture, a crucial finding in treating KS patients for preserving and propagating SSCs for future sperm production, either in vitro or in vivo. This in vitro propagation system can be translated to clinical fertility preservation for KS patients.

Semen preservation by cooling or freezing is an essential artificial insemination (AI) step. AI is affected by many factors, such as semen quality. The aim of this study was to evaluate the effect of adding different concentrations of different antioxidants such as selenium, zinc oxide and Vitamin C to the diluted buck semen during cooling in both summer and winter. Eight mature healthy Zaraibi bucks were used in the study. semen was collected twice weekly during summer 2021 (august and September) and winter 2022 (January and February). Semen samples were collected by an electro- ejaculator device. Samples of good quality semen were pooled and diluted by extender and divided according to experimental groups: Group 1 Vitamin C: 50 mg /100 ml, 1oo mg /100 ml, 150 mg /100 ml. Group 2 Zinc oxide: 0.5 mg /100 ml, 1 mg /100 ml, 1.5 mg /100 ml. Group 3 Selenium: 100 μL/100 ml, 200 μL / 100, 300 μL / 100. After cooling by 1 hr. semen parameters were examined as motility, livability, acrosome integrity and cell membrane integrity and at 8 hours from cooling then every 8 hr till 64 hr. Seminal antioxidant activities as SOD and catalase were estimated at 0 hr, 24 hr and 48 hr. The results indicated that selenium 200 μL / 100 ml diluent has a favorable effect on cooled diluted buck semen during summer and winter more than selenium 100 μL and 300 μL. Selenium is better than Zinc oxide and Vitamin C for improving the semen quality as with minimum cost. In conclusion adding selenium as antioxidant to buck semen during cooling preservation with 200 μL concentration per 100 diluents is preferred for buck semen during cooling preservation. [ABSTRACT FROM AUTHOR]

For animal reproduction, artificial insemination has become a need. Semen preservation has been crucial in artificial insemination as it can determine the success of fertilization and pregnancy. Semen extenders have been critical in prolonging the usability of semen while also retaining its viability, motility, and integrity so that it can be used to fertilize female animals. Due to its nutritional and chemical qualities that can create an environment for spermatozoa to survive and thrive before insemination, coconut water has been the focus of various research addressing its effect on semen preservation. These studies have shown that coconut water has the potential to be an effective semen extender due to its ability to protect the semen during cooling and cryopreservation. Coconut water also includes nutrients (water, sugars, proteins, salts, and vitamins) to help spermatozoa thrive in a healthy environment. However, its performance lags that of commercial extenders, but it can be a less expensive alternative or replacement when commercial extenders are unavailable.

Artificial insemination (AI) appears as a core of reproductive management in the hog industry. AI involves the use of chilled semen doses meeting routine thresholds for motility (≥ 70%) and morphology (≥ 80%) at collection. However, semen doses meeting these standards exhibit a gradual decline in sperm quality, with likely divergent profiles during preserva- tion that impact fertility outcomes. Hence, this study aims to unravel the sperm quality dynamics of boar semen doses during 7-d chilled storage at 17ºC. Semen doses (n = 25) from proven-fertile Duroc boars were provided by a commercial hog farm (Prestage Farm, MS) and transported to the laboratory within 30 min of collection. Each semen sample was aliquoted and analyzed on the day of collection (d 0) and after 7 d of storage (d 7) at 17°C. Sperm total motility (TM), progressive motility (PM), and normal morphology (NM) were assessed using a computer-assisted sperm analyzer (CASA; CEROS II, IMV Biotechnologies). Apoptosis (Annexin V/7-AAD), viability (Propidium iodide – PI), and intracellular reactive oxygen species (ROS; DCFH-DA) were evaluated using flow cytometry. Total antioxidant activity (TAOC) and lipid peroxidation (malondialdehyde; MDA) were assessed using commercial kits (Cayman Chemical). Data analysis was conducted using parametric statistics (SPSS), with P < 0.05 considered significant. Results were pre- sented as mean ± SEM. Data analysis confirmed the significant decrease of CASA parameters, particularly TM from d 0 to d 7 (78.87 ± 1.37 vs. 73.19 ± 1.98%), with extreme variance on d 7 allowing to discriminate high motility (HM, n = 6) vs low motility (LM, n = 6) semen doses. HM doses evidenced significantly greater (P < 0.05) TM (83.73 ± 1.17%), PM (36.07 ± 1.11%), and NM (89.45 ± 2.30%) compared with LM doses (TM: 59.40 ± 3.35%, PM: 16.57 ± 3.25%, NM: 79.93 ± 2.61%) on d 7. Unlike HM samples, which were comparable between d 0 and d 7, LM doses displayed sig- nificantly greater proportions of apoptotic and viable cells, and increased intracellular ROS levels on d 0. Likewise, LM samples revealed significantly greater (P < 0.05) proportions of apoptotic cells (3.69 ± 1.66%), dead cells (38.41 ± 3.71%), and cells with intracellular ROS (36.21 ± 3.62%) than HM samples (apoptotic cells: 0.03 ± 0.02%, dead cells: 4.73 ± 4.65%, ROS: 5.34 ± 4.91%) on d 0; however, only the proportions of dead cells displayed significant increase in LM on d7. MDA concentrations were comparable between HM and LM samples on d 0. However, on d 7, LM samples exhibited significantly greater MDA concentrations than HM samples. In contrast, TAOC was significantly greater in HM samples (2.53 ± 0.14 mM) compared with LM samples (2.17 ± 0.07 mM) on d 0. In conclusion, semen samples with similar gross characteristics at collection exhibited differential responses during storage. Their initial redox potential and apoptosis-like changes may contribute to their varying ability to with- stand prolonged storage. Work supported by USDAARS grant# 6066-31000-015-00D. [ABSTRACT FROM AUTHOR]

Accurate assessment of ram sperm quality is crucial to optimizing assisted reproductive technologies in sheep. However, semen preservation can induce sperm due to osmotic, biochemical, and thermal stress. Stabilizing sperm with a suitable cooling rate and adaptation period to the extender could mitigate these effects for a more reliable evaluation. This study aimed to determine: (1) the best time to assess ram sperm quality, and (2) the factor responsible for the altered state of ram sperm during the first hours of liquid storage. In Experiment 1, ejaculated sperm were diluted and assessed for sperm motility and functionality at four preservation times: 0, 3, 6, and 24 h as sperm damage control. Both sperm motility and functionality improved after 6 h. Experiment 2 investigated the factor responsible for sperm quality change by testing the interactions of seminal plasma and extender with sperm from epididymides independently and in combination. The evaluation of sperm was performed as in Experiment 1. Sperm in groups containing the extender showed altered motility at 0 and 24 h, and lower functionality at 0 h. Thus, we could assume that extender addition initially alters ram sperm, causing sublethal damage that is reversible after 3 to 6 h of semen preservation. In conclusion, ram sperm require an adaptation time of 3 to 6 h to the extender before an accurate quality assessment can be conducted. This has practical implications for reproduction centers, enabling better workflow organization and optimal expression of ram sperm attributes when cervical artificial insemination is routinely performed.

Artificial insemination (AI) is commonly used in the equine industry to enhance the genetic value in breeding programs and to effectively utilize ejaculates. Many stallions are used as breeding stallions as well as in high‐level sports competitions to improve their market value. The goal of the present study was to investigate whether this dual use of stallions influences the animals´ stress levels and/or the quality of their ejaculates. For this purpose, 18 stallions were grouped into two categories: breeding stallions with (BSC = breeding stallion competition), and breeding stallions without secondary use in competitions (BS = breeding stallion). Two ejaculates were collected at a one‐week interval and analysed with an extended spectrum of spermatological methods. Furthermore, saliva, as well as seminal plasma samples were taken, and the concentration of cortisol therein was determined. Additionally, dehydroepiandrosterone (DHEA) and the cortisol/DHEA ratio were analysed and calculated for seminal plasma. After statistical analysis of the correlations and interdependences between the two groups, the results showed that the BSC group had significantly higher saliva cortisol levels (p =.027) and tendentially higher DHEA concentrations in their seminal plasma (p =.056). No difference between BS and BSC could be found in regard to the sperm quality parameters and the cortisol concentration in seminal plasma samples. It can be concluded that while active participation in competitions represents a stress factor, the dual use of stallions in breeding programs and sports competitions is possible without negative effects on their sperm quality. [ABSTRACT FROM AUTHOR]

Multi-drug antibiotic resistance of Serratia (S.) marcescens and Klebsiella (K.) oxytoca in boar semen is an emerging threat to pig reproduction and the environment. The aim of this study is to examine the efficiency of a novel hypothermic preservation method to inhibit the growth of these bacterial species in extended boar semen and to maintain the sperm quality. The semen samples extended in an antibiotic-free Androstar Premium extender were spiked with ~102 CFU/mL of S. marcescens or K.oxytoca. Storage at 5 °C for 144 h inhibited the growth of both bacterial species and maintained the sperm quality, whereas bacterial counts increased to more than 1010 CFU/mL in the 17 °C samples used as positive controls. This was accompanied by an increase in the sperm agglutination and the loss of motility and membrane integrity. We conclude that hypothermic storage is a promising tool to combat resistant bacteria in boar semen and to contribute to the One Health approach. [ABSTRACT FROM AUTHOR]

Background: Semen quality and related parameters correlate directly with fertilization, consequently assisting reproductive technology outcomes. Traditional studies on carob (Ceratonia siliqua) have demonstrated its effect on male fertility potential via the reductive effect on reactive oxygen species. This study aimed to investigate the effect of carob kibble extract on sperm motility. Methods: The extract was made using acetone as a solvent, followed by vacuum evaporation and filtration. Following thawing, each of the forty human semen samples was divided into four groups and exposed to concentrations of 0.0 (Control), 0.05, 0.2, and 0.8 mg/ml of the extract. Percentages of progressive motile, nonprogressive motile, and immotile sperms, as well as other kinematic parameters, were assessed by computer-aided sperm analysis immediately after exposure to the concentrations (T0) and one hour later (T1). Data were analyzed by repeated measure analysis of variance and paired sample t-student tests using SPSS software. The level of p< 0.05 was considered statistically significant. Results: No significant difference was found between groups at T0 or T1 values. However, a comparison of matched doses at T0 and T1 indicated that lower doses 0.05 and 0.2 mg/ml could significantly (p<0.05) inhibit natural decline in motility. Conclusion: Adding lower doses of carob kibble extract on a thawing medium could have a supportive effect on sperm motility. However, adding the extract to a vitrification solution before a freezing process, as well as oral intake of the extract seems to have more efficiency than would be a subject for further studies. [ABSTRACT FROM AUTHOR]

The aim of this study was first to examine the prevalence of bacteria-associated loss of sperm quality in samples from insemination centers during a seven-year semen monitoring program and, second, to investigate the growth dynamic of four different multidrug-resistant bacterial species and their impact on sperm quality during semen storage. A reduced sperm quality associated with bacterial contamination was found in 0.5% of 3219 of the samples from insemination centers. In samples spiked with Serratia marcescens and Klebsiella oxytoca, bacterial growth by six log levels was seen during storage at 17 °C, causing loss of sperm motility, membrane integrity, membrane fluidity, and mitochondrial membrane potential at >107 CFU/mL (p < 0.05). Storage at 5 °C in the Androstar Premium extender efficiently inhibited their growth. Achromobacter xylosoxidans and Burkholderia cepacia showed limited growth up to two log levels at 17 °C and did not impair sperm quality. In conclusion, spermatozoa tolerate moderate loads of multidrug-resistant bacteria, and hypothermic, antibiotic-free semen storage effectively limits bacterial growth. The constant use of antibiotics in semen extenders should be reconsidered. [ABSTRACT FROM AUTHOR]

Background and objectives: Semen cryopreservation is widely used in assisted reproduction techniques, and reliable semen analysis is essential to define the clinical practice. However, many parameters used for semen evaluation have high variability among technicians. Here, we describe a method of validating semen analysis prior to cryopreservation, comparing each operator’s results with an expert, and also analyzing inter-operator variability. As a second endpoint, we compare this method by analyzing semen parameters before and after cryopreservation. Methods: Four professional trainees studied and practiced semen analysis according to the World Health Organization guidelines for one month, under supervision of an expert in the field. Next, microscopic results (sperm concentration, motility, vitality, and morphology) obtained by each team member were compared with the findings obtained by the expert.. Finally, analyzes of inter-operators were evaluated for the same parameters. Results: The findings obtained by the operators and the expert did not differ significantly. Furthermore, in the inter-operator analysis, the morphology parameter differed significantly in the fresh semen sample, which was not observed in the post-thaw sample. Conclusion: Our results indicated that the laboratory staff training for semen analysis was effective, ensuring the assessment of individual performance and uniformity among operators in sperm count parameters, producing consistent results.

Storage of boar semen at 5 °C instead of the conventional temperature of 17 °C is an innovative preservation concept. It enhances protection against the growth of bacteria normally occurring in the ejaculates and potential drug-resistant contaminants from the environment. Thereby it allows the reduction or even elimination of antibiotics in porcine semen extenders. The present article reviews the current state of the low-temperature preservation approach of boar semen, with a special focus on antimicrobial efficiency and fertility in field insemination trials. Particularly the role of semen extenders and temperature management for the achievement of high fertility and biosecurity are elucidated. Insemination data of 1,841 sows in there different countries revealed equally high farrowing rates and litter sizes of semen stored at 5 °C compared to the controls stored at 17 °C. Microbiology data obtained from semen doses spiked with multi-drug resistant bacteria showed the efficiency of the cold semen storage for inhibiting the growth of Serratia marcescens , a bacterial species with high sperm-toxicity. Evolving concepts on the physiological role of the male reproductive microbiome for female fertility provides a further argument against the complete eradication of bacteria in the semen dose by antibiotic additives to the extenders. Finally, motivation and practical considerations for the use of the novel preservation tool in artificial insemination of pigs are revealed, which might encourage the transformation towards a sustainable production of boar semen doses following the One Health approach. • Boar semen storage at 5 °C is an innovative tool to reduce antibiotics in extenders. • Field data from 1,841 inseminated sows show high fertility with 5 °C semen. • The 5 °C semen storage is effective against drug resistant Serratia marcescens. • Holding at 17 °C before cooling improves sperm quality and is bio-safe. • The 5 °C semen storage is ready-to-use in pig insemination. [ABSTRACT FROM AUTHOR]

Background: Prolonging the shelf‐life of liquid‐preserved semen without compromising its fertilizing capacity may increase the efficiency of artificial insemination in pigs. Many fertilization‐relevant processes are adenosine triphosphate dependent. The impact of semen storage and rewarming to body temperature on the energy status of spermatozoa is as yet unknown. Objectives: To investigate the energy status of boar spermatozoa during storage and subsequent rewarming and to reveal the potential role of mitochondrial function for reactivation and maintenance of sperm motility. Materials and methods: Extended semen samples (n = 7 boars) were used. Spermatozoa were challenged by storage at 17°C for 7 days and incubation at 38°C for 180 min. The adenosine triphosphate concentration and energy charge in semen samples and lactate concentration in the extracellular medium were assessed. Viability and mitochondrial activity were determined by flow cytometry, and clustered single‐cell analysis of motility parameters was performed. Results: The energy status was not affected by semen storage (p > 0.05). Rewarming resulted in a net reduction in adenosine triphosphate concentration, which increased with storage time (maximum Day 5: ‐24.2 ± 10.3%) but was not accompanied by a loss in viability, motility, or mitochondrial activity. Blocking glycolysis with 2‐deoxy‐d‐glucose prevented the re‐establishment of motility and mitochondrial activity after rewarming. Mitochondrial activity gradually subsided in virtually all spermatozoa during incubation at 38°C, while adenosine triphosphate and energy charge remained high. Concomitantly, extracellular lactate levels rose, and sperm populations with lower velocity, increased linearity, and low lateral head displacement grew larger. Size changes for major sperm subpopulations correlated with the percentage of viable spermatozoa with high mitochondrial activity (r = 0.44–0.70 for individual subpopulations, p < 0.01). Conclusion: Storage of boar spermatozoa increases the demand of adenosine triphosphate for reactivation of spermatozoa towards fast, non‐linear, and hyperactivation‐like motility patterns upon rewarming. Maintenance of glycolysis seems to be decisive for sperm function after long‐term storage in vitro. [ABSTRACT FROM AUTHOR]

Collection of epididymal stallion sperm offers the opportunity to retain and use genetic material from the males after elective castration or even post mortem. The aim of the present study was to evaluate the effect of the Ringer B. Braun solution on stallion epididymal sperm viability and motility during liquid storage for 72 h at 4 °C and at 20 °C. Eight stallions (3–10 years old) were used in the study. Sperm from the cauda epididymis was harvested immediately after routine castration. The sperm from epididymis was washed out and diluted with Ringer B. Braun solution and with a commercial semen extender which was used as a control. Samples from each horse were divided into two parts: one part of samples was stored at 4 ± 1 °C as recommended for the commercial semen extender, the other one was stored at 20 ± 1 °C. Sperm viability, motility, and pH were checked one hour after collection and dilution, and after 24, 48 and 72 h of incubation. The results of sperm viability, subjective and progressive sperm motility showed slightly higher results in semen samples diluted with Ringer B. Braun solution in one hour compared to the commercial extender, by 2.40 ± 0.49% for viability (P > 0.05), by 0.30 ± 9.92% for subjective motility (P > 0.05) and by 5.70 ± 6.07% for progressive motility (P > 0.05). We suggest that Ringer solution could be used for a short term storage (1–24 h) of equine epididymal semen at a 4 °C temperature. [ABSTRACT FROM AUTHOR]

The aim was to assess the reproductive efficiency of different techniques used to preserve spermatozoa in artificial insemination semen doses (AI‐doses) by evaluating refrigeration at 15°C, cryopreservation and encapsulation. Forty‐two hyperprolific sows were treated with buserelin and inseminated once at a single fixed time. The fertility rate, embryonic vesicles viability and the early embryonic mortality (arrested conceptuses) evaluated post‐mortem at 24th day of pregnancy, were analysed in order to assess the effectiveness of each proposed technique. Results show an overall reduction on fertility using the three proposal sperm preservation techniques (69.27%, 60.00% and 78.75% for refrigerated, frozen–thawed and encapsulated AI‐doses, respectively). Total number of embryonic vesicles was very similar among the three treatments; yet, the number of viable vesicles was numerically different among groups, and thus, embryonic viability was 79.25%, 80.0% and 87.15% for refrigerated, frozen–thawed and encapsulated AI‐doses, respectively. [ABSTRACT FROM AUTHOR]

Background and objectives: Semen cryopreservation is widely used in assisted reproduction techniques, and reliable semen analysis is essential to define the clinical practice. However, many parameters used for semen evaluation have high variability among technicians. Here, we describe a method of validating semen analysis prior to cryopreservation, comparing each operator’s results with an expert, and also analyzing interoperator variability. As a second endpoint, we compare this method by analyzing semen parameters before and after cryopreservation. Methods: Four professional trainees studied and practiced semen analysis according to the World Health Organization guidelines for one month, under supervision of an expert in the field. Next, microscopic results (sperm concentration, motility, vitality, and morphology) obtained by each team member were compared with the findings obtained by the expert. Finally, analyzes of inter-operators were evaluated for the same parameters. Results: The findings obtained by the operators and the expert did not differ significantly. Furthermore, in the interoperator analysis, the morphology parameter differed significantly in the fresh semen sample, which was not observed in the post-thaw sample. Conclusion: Our results indicated that the laboratory staff training for semen analysis was effective, ensuring the assessment of individual performance and uniformity among operators in sperm count parameters, producing consistent results. [ABSTRACT FROM AUTHOR]

Background: The aim of this study was to compare the quality of bull sperm produced in 0.5 and 0.25 ml semen straws before and after freezing. Methods: In this experimental study, semen samples were collected from 12 dual purpose Simmental (Fleckvieh) bull kept at Iran Simmental Cattle Breeding Center during a two-month period. Each ejaculate was divided into two equal portions. Then, the formulation and freezing processes were performed separately for each type of straw. Sperm qualitative characteristics, antioxidant and oxidant levels of frozen semen produced in bulls' 0.5 and 0.25 were compared. Results: The results showed that all sperm quality parameters in 0.25 ml straws were better than 0.5 ml before and after freezing. Catalase (CAT) activity was higher in 0.5 ml straws than 0.25 one, but it was not statistically significant. Malondialdehyde (MDA) levels were lower in 0.5 ml straws than 0.25 ml straws, but the differences were not statically significant. Moreover, no remarkable difference was observed in the activity of superoxide dismutase (SOD) activity enzyme. Conclusion: Based on the findings of the present study and with the aim of reducing the production costs, it is recommended that bull sperm production centers and herdsman inseminators use 0.25 ml straws for semen freezing and cattle artificial insemination. [ABSTRACT FROM AUTHOR]

Multi-drug antibiotic resistance of Serratia (S.) marcescens and Klebsiella (K.) oxytoca in boar semen is an emerging threat to pig reproduction and the environment. The aim of this study is to examine the efficiency of a novel hypothermic preservation method to inhibit the growth of these bacterial species in extended boar semen and to maintain the sperm quality. The semen samples extended in an antibiotic-free Androstar Premium extender were spiked with ~102 CFU/mL of S. marcescens or K.oxytoca. Storage at 5 °C for 144 h inhibited the growth of both bacterial species and maintained the sperm quality, whereas bacterial counts increased to more than 1010 CFU/mL in the 17 °C samples used as positive controls. This was accompanied by an increase in the sperm agglutination and the loss of motility and membrane integrity. We conclude that hypothermic storage is a promising tool to combat resistant bacteria in boar semen and to contribute to the One Health approach.

Loquat leaves are the by-product of loquat fruit production. Polysaccharides are one of the main active ingredients in loquat leaves. In this study, polysaccharides were extracted from loquat leaves by ultrasonic-assisted deep eutectic solvents (DESs) extraction method. Further, the extracted crude loquat leaf polysaccharides (CLLP) were purified and separated via S-8 resin and DEAE-52 cellulose column chromatography, respectively. Additionally, the effects of polysaccharides on activity of sperm in boar semen preserved in medium at 17 °C, were evaluated preliminarily. DES, composed of choline chloride/ethylene glycol (1:6, molar ratio), was proved to be the suitable solvent for LLP extraction. The optimized extraction conditions were water content 44 %, liquid-solid ratio 1:29 (g/g), extraction temperature 61 °C and extraction time 98 min. Under these conditions, the LLP yield was 57.82 ± 1.50 mg/g. A homogeneous polysaccharide (LLP1-2, M w : 2.17 × 104 Da) was isolated from CLLP. Its total sugar, uronic acid and protein contents were 76.31 ± 1.25 %, 14.19 ± 0.67 % and 3.28 ± 0.42 %, respectively. Further, 800 μg/mL LLP1-2 could effectively enhance the antioxidant activity of sperm. This study laid a foundation for DESs and column chromatography in the field of polysaccharide extraction and separation, proving that LLP can be used as a natural antioxidant for sperm preservation. • Choline chloride/ethylene glycol is a promising solvent for LLP extraction. • S-8 resin and DEAE-52 cellulose were employed for LLP purification and separation. • Lower molecular weight and higher glucuronic acid content LLP was obtained. • LLP is a promising natural exogenous antioxidant for boar semen preservation. [ABSTRACT FROM AUTHOR]

The aim of this study was first to examine the prevalence of bacteria-associated loss of sperm quality in samples from insemination centers during a seven-year semen monitoring program and, second, to investigate the growth dynamic of four different multidrug-resistant bacterial species and their impact on sperm quality during semen storage. A reduced sperm quality associated with bacterial contamination was found in 0.5% of 3219 of the samples from insemination centers. In samples spiked with Serratia marcescens and Klebsiella oxytoca, bacterial growth by six log levels was seen during storage at 17 °C, causing loss of sperm motility, membrane integrity, membrane fluidity, and mitochondrial membrane potential at >107 CFU/mL (p < 0.05). Storage at 5 °C in the Androstar Premium extender efficiently inhibited their growth. Achromobacter xylosoxidans and Burkholderia cepacia showed limited growth up to two log levels at 17 °C and did not impair sperm quality. In conclusion, spermatozoa tolerate moderate loads of multidrug-resistant bacteria, and hypothermic, antibiotic-free semen storage effectively limits bacterial growth. The constant use of antibiotics in semen extenders should be reconsidered.

Objective. Evaluate the effect of Bovine Serum Albumin (BSA) concentration on the freezability of goat semen in a Tris-based lyophilized preservative-diluter, without performing seminal washing, compared with a control preservative-diluter of lactose-skimmed milk (DC), removing the seminal plasma by centrifugation. Materials and methods. It will cover 90 ejaculates, volume, motility, concentration, viability, and sperm morphology. The fit ejaculates were mixed and the pool divided into five portions, each receiving one of 4 lyophilized combinations based on Tris-Glucose-Ac. Citrus and Glycerol with different concentrations of BSA (0.1%, 0.5%, 1% and 2%) or DC. It was frozen in 0.1 ml pellets in nitrogen vapors, and after 2 min., They were stored in liquid nitrogen until thawing 7 days later, the following were determined: motility percentage (30, 120 and 240 minutes), viability, damaged acrosomes and total anomalies (30 and 120 minutes) and were compared using a Binary Logistic Regression model. Results. The highest sperm motility and viability (p<0.05) in the three times was for 0.5%, 1% and 2% of BSA, which were higher than 0.1% of BSA and DC. Damages acrosome and total abnormalities at 30 and 120 minutes were lower (p<0.05) for 0.5%, 1% and 2% of BSA compared to 0.1% and DC. Conclusions. The cryopreservation of goat semen does not require seminal washing by centrifugation if 0.5-2% BSA is used as a membrane protector in a lyophilized dilute-conservative based on Tris-Glucose-Citric Acid and Glycerol. [ABSTRACT FROM AUTHOR]

Abstract Background Hypothermic preservation of boar semen is considered a potential method for omitting antibiotics from insemination doses, thereby contributing to the global antibiotic resistance defence strategy. The main challenges are chilling injury to spermatozoa and bacterial growth during semen storage leading to reduced fertility. Objectives To examine chilling injury and the number and type of bacteria in boar semen stored at 5 °C in the absence of antibiotics, and to assess the applicability of hypothermic semen storage under field conditions. Material and methods Boar ejaculates were extended with AndroStar® Premium, stored at 17 °C with and at 5 °C without antibiotics and tested for functional sperm parameters by flow cytometry. Raw semen and extended samples were investigated bacteriologically. Fertility was evaluated after once-daily inseminations of 194 sows in a field study. Results Lethal sperm damage assessed by motility and membrane integrity was low throughout storage in both experimental groups. Sublethal chilling effects based on the decrease of viable spermatozoa with low membrane fluidity were higher (P 0.05) between sow groups inseminated with semen stored antibiotic-free at 5 °C and semen stored at 17 °C with antibiotics. Conclusion Despite subtle chilling effects and low bacterial numbers, antibiotic-free hypothermic storage of boar semen offers the possibility to reduce the use of antibiotics in pig insemination. However, strict sanitary guidelines must be maintained and further evidence of efficiency under field conditions is considered desirable.

Background: Many cancer treatments pose a threat to fertility for patients. Semen cryopreservation before cancer treatment is an effective method to preserve fertility. There are sparse long-term data on the usage of samples from Canadian oncology sperm banks., Methods: A retrospective chart review of all oncology sperm banking samples at a Canadian academic fertility centre from 2001 to 2020 was conducted., Results: From 2001 to 2020, 4521 samples were banked by 2504 patients. The most frequent diagnoses among these patients were testicular cancer (29.5%) and lymphoma (26.9%). Of these patients, only 81 (3.2%) patients returned to use their samples with intrauterine insemination (IUI) or in vitro fertilisation (IVF) treatment and 62 (2.5%) patients transferred their samples to another clinic. The time between banking and return for usage of the sperm ranged from 1 to 131 months with a median of 18 months after banking. A total of 66 IVF cycles (104 embryo transfers) and 101 IUI cycles from 67 patients were reviewed. Of the 67 couples who used their samples, 53.7% achieved a clinical pregnancy. The clinical pregnancy rate was 6.6% per cycle for IUI and 30.8% per embryo transfer for IVF. Higher sperm concentration or total motile count was not associated with a higher chance of pregnancy. Patients who conceived had on average 1.9 ± 0.8 (p=0.02) more usable embryos per cycle than those who did not conceive., Conclusions: Sperm cryopreservation provides a valuable option for patients with cancer to achieve parenthood after potentially gonadotoxic cancer treatment. However, the overall usage of banked oncology sperm samples is very low., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose: To investigate cryopreserved testicular spermatozoa among patients with azoospermia., Methods: In this retrospective study spanning from October 1993 to December 2021, we examined men diagnosed with azoospermia who underwent testicular spermatozoa cryopreservation. Data from medical records included utilization and disposal of sperm samples, age at initial cryopreservation. We analyzed the data over 20 years using Kaplan-Meier curves, compared age with the log-rank test, and assessed hazard ratios (HR) with 95% confidence intervals (CI) using Cox regression analysis., Results: A total of 356 patients with a mean age of 32.1 ± 6 were included. Of these, 225 patients utilized thawed testicular sperm for fertility treatments, with 118 patients using all their frozen straws and 107 patients partially using their stored straws. Additionally, 29 patients opted for disposal (six patients partially used their testicular spermatozoa before disposal), resulting in 108 patients who neither used nor disposed of their straws. From a laboratory standpoint, nearly 90% of patients contributed a single testicular sample, which was subsequently divided and cryopreserved as straws, with a median of 4 straws per sample. Notably, in the older age group (> 35 years old), there were a significantly lower usage rate and a higher disposal rate compared to the younger age groups (p < 0.05 for both), corroborated by univariable Cox analysis., Conclusions: This extensive study unveils unique patterns in the preservation and disposal of testicular spermatozoa among azoospermic patients. Most patients utilize a significant portion of their stored samples, while older patients tend to use their testicular spermatozoa less frequently., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The shipping of liquid preserved semen is common practice in animal breeding and prior to cryopreservation for gene banking. Vibration emissions during transport may be harmful to spermatozoa. Therefore, strategies to minimize agitation-induced sperm injury are needed. The aim was to examine whether the type of semen extender, time after semen processing and the temperature in simulated transport conditions influence the response of boar spermatozoa to agitation stress. In Experiment 1, boar semen samples (n = 16) extended in Beltsville Thawing Solution (BTS) or Androstar Plus (APL) medium were filled in 90 mL tubes and shaken for 4 h at 200 rpm either at 22 °C or 17 °C. Samples were then stored at 17 °C for 144 h. In Experiment 2, semen samples (n = 11) extended in Androstar Premium were shaken either directly after filling at 22 °C or 20 h later after cooling to 5 °C. Samples were stored at 5 °C for 144 h. In Experiment 1, sperm motility and viability were lower (p < 0.05) in the shaken samples compared to the controls. The temperature, extender and the storage length had no effect on the agitation-induced loss of sperm quality. Sperm quality traits were higher in samples stored in APL compared to BTS. In Experiment 2, sperm motility at 24 h was reduced (p < 0.05) in those samples shaken at 22 °C but not at 5 °C. Sperm viability, membrane fluidity and mitochondrial membrane potential were not affected in either of the treatment groups. Extended boar semen designed for 17 °C storage and shipped on the day of collection is sensitive to agitation stress. In contrast, spermatozoa slowly cooled to 5 °C and shaken 20 h after processing are more resistant to agitation-induced shear forces and interfacial phenomena. • Agitation stress simulating shipping of freshly processed boar semen doses reduces sperm quality. • Semen cooled to 5 °C overnight is less sensitive to agitation stress at 5 °C. • The agitation effects on sperm quality are not induced by pH shifts in the extended semen. • The response to agitation stress does not affect the long-term preservability of extended semen. • Strategies to reduce shear forces and interfacial phenomena during shipping are proposed. [ABSTRACT FROM AUTHOR]

This review reports the current status of artificial breeding technology in the Crocodylia and the future requirements for the establishment of AI in the saltwater crocodile. Although there are challenges regarding safe restraint and immobilisation, semen collection of the saltwater crocodile by manual stimulation has proven effective in yielding sufficient volume and sperm concentrations for empirical and molecular analyses of sperm preservation and physiology. Nevertheless, there is still much to learn with respect to fundamental anatomy, physiology and behaviour in both sexes, but particularlyin the female. Althoughlessons canbe learned from successful AI inthe alligator, the details of this researcharenot readily accessible. Future research needs to focus on the proximate factors of seasonality and the underlying control of the female's annual reproductive cycle; thiswill require novel and innovativeways tocollect blood sampleswithout causing stress or injury, and ideally a dedicated crocodile research breedingcolony. Because the saltwater crocodile is a farmed species, there is likely to be sufficient impetus for the application of assisted breeding technology to drive future productivity in the industry. These developmentswill also have benefits for the genetic and reproductivemanagement of endangered captive populations. [ABSTRACT FROM AUTHOR]

Background: Klinefelter’s Syndrome (KS) is rarely reported in China, which is associated with insufficient sperm production and infertility in male patients. Despite the fact that infertility treatments, such as intracytoplasmic sperm injection (ICSI) and preimplantation genetic diagnosis (PGD), have been widely used in KS population, almost all these patients have to use donor semen in China to establish successful pregnancies nowadays. Case Report: The authors report a case of KS in a patient with unremarkable characteristics except for oligospermia. The patient presented to this center with infertility and the chromosome analysis demonstrated a non-mosaic 47, XXY karyotype. Further testing showed no deletions in the SRY, AZF-a, AZF-b, and AZF-c genes. Finally, the patient successfully impregnated the partner, and then the partner delivered a healthy male neonate. The patient became fertile through ICSI, together with cryopreservation of a small number of spermatozoa after the first blastocyst transfer. Conclusion: This report further confirms that KS men can father their own healthy children. While adequate sperm cryopreservation and blastocyst transfers are strongly recommended. Nevertheless, it is necessary for such couples to be offered extensive genetic counseling before pregnancy and prenatally.

Sperm become most sensitive to cold shock when cooled from 37 °C to 5 °C at rates that are too fast or too slow; cold shock increases the susceptibility to oxidative damage owing to its influence on reactive oxygen species (ROS) production, which are significant stress factors generated during cooling and low temperature storage. In addition, ROS may be a main cause of decreased motility and fertility upon warming. They have been shown to change cellular function through the disruption of the sperm plasma membrane and through damage to proteins and DNA. The objective of this study was to determine which cryopreservation rates result in the lowest degree of oxidative damage and greatest sperm quality. In the rhesus model, it has not been determined whether suprazero cooling or subzero freezing rates causes a significant amount of ROS damage to sperm. Semen samples were collected from male rhesus macaques, washed, and resuspended in TEST-yolk cryopreservation buffer to 100 × 10(6) sperm/mL. Sperm were frozen in 0.5-mL straws at four different combinations of suprazero and subzero rates. Three different suprazero rates were used between 22 °C and 0 °C: 0.5 °C/min (slow), 45 °C/min (medium), and 93 °C/min (fast). These suprazero rates were used in combination with two different subzero rates for temperatures 0 °C to -110 °C: 42 °C/min (medium) and 87 °C/min (fast). The different freezing groups were as follows: slow-med (SM), slow-fast (SF), med-med (MM), and fast-fast (FF). Flow cytometry was used to detect lipid peroxidation (LPO), a result of ROS generation. Motility was evaluated using a computer assisted sperm motion analyzer. The MM and FF treated sperm had less viable (P < 0.0001) and motile sperm (P < 0.001) than the SM, SF, or fresh sperm. Sperm exposed to MM and FF treatments demonstrated significantly higher oxidative damage than SM, SF, or fresh sperm (P < 0.05). The SM- and SF-treated sperm showed decreased motility, membrane integrity, and LPO compared with fresh semen (P < 0.001). Slow cooling from room temperature promotes higher membrane integrity and motility post thaw, compared with medium or fast cooling rates. Cells exposed to similar cooling rates with differing freezing rates were not different in motility and membrane integrity, whereas comparison of cells exposed to differing cooling rates with similar freezing rates indicated significant differences in motility, membrane integrity, and LPO. These data suggest that sperm quality seems to be more sensitive to the cooling, rather than freezing rate and highlight the role of the suprazero cooling rate in post thaw sperm quality.

BackgroundModification of cryoprotective medium (CPM) R18S3 (18% raffinose and 3% skim milk) by addition of monothioglycerol (MTG) or L-glutamine (Glu) has been shown to improve in vitro fertilization (IVF) using mouse sperm cryopreserved in cryostraws. However, whether these CPMs can be applied effectively to sperm cryopreserved in cryovials is unknown.ObjectiveThe study was to determine the comparative effectiveness of using R18S3, R18S3+Glu (100mM and 87 mM), or R18S3+MTG (477 µM) to cryopreserve various sample volumes of mouse sperm in cryovials and cryostraws.MethodsThis study compared the effects of different CPMs on motility of fresh and frozen-thawed C57BL/6J sperm and on IVF rate of C57BL/6J sperm cryopreserved in different CPMs and containers with different volumes, and then used technologies developed to cryopreserve and recover sperm of knockout mouse lines on inbred C57BL/6 backgrounds.ResultsGlutamine at 100 mM inhibited, but MTG at 477 µM protected, fresh sperm motility significantly (P < 0.05). Sperm cryopreserved in R18S3+MTG had significantly better (P < 0.05) post-thaw progressive motility and IVF rate than when cryopreserved in R18S3 alone, R18S3+Glu (100 mM), or RSGlu87 (15.7% raffinose, 2.6% skim milk, and 87 mM L-glutamine). There was no significant difference in IVF rates among sperm cryopreserved with R18S3+MTG in cryovials or in cryostraws (P > 0.05). Sperm from 63 knockout mouse lines on C57BL/6 backgrounds cryopreserved using R18S3+MTG in cryovials were all recovered successfully to genotypically-confirmed offspring.ConclusionMouse sperm on C56BL/6 backgrounds can be successfully cryopreserved in cryovials using R18S3+MTG.

Abstract We report the carboxylated C60 improved the survival and quality of boar sperm during liquid storage at 4 °C and thus propose the use of carboxylated C60 as a novel antioxidant semen extender supplement. Our results demonstrated that the sperm treated with 2 μg mL−1 carboxylated C60 had higher motility than the control group (58.6% and 35.4%, respectively; P ˂ 0.05). Moreover, after incubation with carboxylated C60 for 10 days, acrosome integrity and mitochondrial activity of sperm increased by 18.1% and 34%, respectively, compared with that in the control group. Similarly, the antioxidation abilities and adenosine triphosphate levels in boar sperm treated with carboxylated C60 significantly increased (P ˂ 0.05) compared with those in the control group. The presence of carboxylated C60 in semen extender increases sperm motility probably by suppressing reactive oxygen species (ROS) toxicity damage. Interestingly, carboxylated C60 could protect boar sperm from oxidative stress and energy deficiency by inhibiting the ROS-induced protein dephosphorylation via the cAMP-PKA signaling pathway. In addition, the safety of carboxylated C60 as an alternative antioxidant was also comprehensively evaluated by assessing the mean litter size and number of live offspring in the carboxylated C60 treatment group. Our findings confirm carboxylated C60 as a novel antioxidant agent and suggest its use as a semen extender supplement for assisted reproductive technology in domestic animals.

Purpose: Sperm cryopreservation before cancer treatment is the most effective method to preserve the fertility of male pa-tients. We present our 21 years experience with sperm cryopreservation for cancer patients, including an examination of se-men quality, the current status of cryopreserved sperm, and the rate of sperm use for assisted reproductive technology (ART). Materials and Methods: A total of 721 cancer patients at Fertility Center of CHA Gangnam Medical Center successfully per-formed sperm cryopreservation for fertility preservation from January 1996 to December 2016. Medical chart review was used to analyze patient age, marital status, cancer type, semen volume, sperm counts and motility, length of storage, and cur-rent banking status. Results: The major cancers of the 721 patients were leukemia (28.4%), lymphoma (18.3%), testis cancer (10.0%). The mean age at cryopreservation was 27.0 years, and 111 patients (15.4%) performed sperm cryopreservation during or after cancer treatment. The mean sperm concentration was 66.7±66.3 ×106/mL and the mean sperm motility was 33.8%±16.3%. During median follow-up duration of 75 months (range, 1–226 months), 44 patients (6.1%) used their banked sperm at our fertility center for ART and 9 patients (1.2%) transferred their banked sperm to another center. The median duration from cryopreser-vation to use was 51 months (range, 1–158 months). Conclusions: Sperm cryopreservation before gonadotoxic treatment is the most reliable method to preserve the fertility of male cancer patients. Sperm cryopreservation should be offered as a standard of care for all men planning cancer therapy.

Developing effective cooled semen protocols is essential to increase pregnancy rates and reproductive efficiency in donkeys. This study aimed to evaluate the effect on sperm kinetic parameters and membrane integrity in cooled donkey semen diluted with defined milk proteins extender with 1% or 2% of egg yolk and the removal of seminal plasma. Twenty-four ejaculates from six jackasses were collected. Each ejaculate was divided into four aliquots that were diluted in extender with 1% (EY1) or 2% (EY2) egg yolk. One sample from each group was centrifuged, seminal plasma was removed (CEY1, CEY2 groups, respectively), and the samples were then refrigerated at 5 °C for 24 h. Fresh and cooled semen samples were assessed for sperm motility, morphology, and plasma membrane integrity. Total motility, progressive motility, sperm kinetic parameters, or live sperm cells were not statistically different when semen was cooled with an extender supplemented with 1% or 2% of egg yolk. Seminal plasma removal does not affect total motility or sperm kinetic parameters. However, progressive motility decreased (P

Sensitive detection of chilling injury in boar spermatozoa is required to evaluate novel hypothermic preservation concepts. The study’s aim was to examine whether analyses of motility patterns and sperm binding in a competitive oviduct explant assay (cOEA) sensitively detect chilling-induced alterations in sperm function. Semen samples (n = seven boars) were split into four subsamples by dilution either in Beltsville Thawing Solution (BTS) or Androstar® Plus and stored at 5 °C or 17 °C. Storage temperature had a significant effect on the distribution of spermatozoa in seven major kinematic clusters. The effect size of chilling at 5 °C as estimated by Cramer’s V was higher (p < 0.05) in the BTS medium (0.21) compared to AndroStar® Plus (0.11). Spermatozoa extended in Androstar® Plus had higher relative binding capacity compared to sperm in BTS (p < 0.05). Binding indices correlated with the percentage of viable, acrosome-intact (r = 0.62) and motile spermatozoa (r = 0.72, both p < 0.001). The cluster size of sperm with slow, vigorous movement was negatively correlated with sperm-oviduct binding (r = −0.43, p < 0.05). In conclusion, the cluster analysis of sperm kinematics and competitive sperm oviduct binding in vitro present meaningful biological tests to assess novel concepts for hypothermic semen preservation.

Background: Hypothermic preservation of boar semen is considered a potential method for omitting antibiotics from insemination doses, thereby contributing to the global antibiotic resistance defence strategy. The main challenges are chilling injury to spermatozoa and bacterial growth during semen storage leading to reduced fertility. Objectives: To examine chilling injury and the number and type of bacteria in boar semen stored at 5 °C in the absence of antibiotics, and to assess the applicability of hypothermic semen storage under field conditions. Material and methods: Boar ejaculates were extended with AndroStar® Premium, stored at 17 °C with and at 5 °C without antibiotics and tested for functional sperm parameters by flow cytometry. Raw semen and extended samples were investigated bacteriologically. Fertility was evaluated after once-daily inseminations of 194 sows in a field study. Results: Lethal sperm damage assessed by motility and membrane integrity was low throughout storage in both experimental groups. Sublethal chilling effects based on the decrease of viable spermatozoa with low membrane fluidity were higher (P < 0.05) up until 72 h in sperm stored at 5 °C compared to 17 °C but did not differ after 144 h. After 72 h, incubation in capacitating medium for 60 min induced a similar decrease in viable sperm with high mitochondria membrane potential and low cytosolic calcium in both groups. In semen stored at 5 °C, bacteria counts were below 103 CFU/mL and the bacteria spectrum was similar to that of raw semen. In 88% of 34 boars, cooled semen fulfilled the requirements for insemination. Fertility was high and did not differ (P > 0.05) between sow groups inseminated with semen stored antibiotic-free at 5 °C and semen stored at 17 °C with antibiotics. Conclusion: Despite subtle chilling effects and low bacterial numbers, antibiotic-free hypothermic storage of boar semen offers the possibility to reduce the use of antibiotics in pig insemination. However, strict sanitary guidelines must be maintained and further evidence of efficiency under field conditions is considered desirable. [ABSTRACT FROM AUTHOR]

Efficient artificial insemination (AI) with liquid preserved boar semen is essential for the swine industry. Glucose is the most widely utilized energy source in refrigerated boar semen extenders. However, the relationship between glucose concentration and spermatozoa quality is not fully understood. In the present study, boar spermatozoa were used as a model to investigate the impact of different glucose concentrations on spermatozoa motility, mitochondrial activity, and acrosome integrity at physiological temperature (37 °C) and during refrigeration (17 °C). The proportion of progressively motile spermatozoa and mitochondrial activity in the high glucose group were significantly lower than in the low glucose group when incubated at 37 °C for 3 h or 17 °C for 3 d, but not at 17 °C for 7 d. Lysine acetylation is a reversible post-translational modification that plays a crucial role in spermatozoa function. Our results show that spermatozoa protein acetylation levels were higher in the high glucose group than in the low glucose group. The proportions of progressively motile and acrosome-intact spermatozoa were higher in acetyltransferase inhibitor (WM-1119)-treated spermatozoa than in the control. Spermatozoa acetyl-CoA concentration, which is directly linked to acetylation, was significantly higher in the high glucose group than in the low glucose group. Taken together, spermatozoa motility and acrosome integrity can be altered by changing the concentration of glucose in the extender. High glucose concentration-induced lysine acetylation participates in the regulation of boar spermatozoa motility and acrosome integrity during preservation. These results can provide insights into spermatozoa preservation and AI in the swine industry. • Boar sperm motility were lower in high glucose condition than low glucose condition. • High glucose concentration induced increase in protein lysine acetylation in sperm. • Inhibition of lysine acetylation promotes sperm motility and delays acrosome reaction. [ABSTRACT FROM AUTHOR]

Sperm cryopreservation decreases motility, probably due to changes in protein phosphorylation. Our objective was to use quantitative phosphoproteomics for systematic comparative analyses of fresh versus frozen-thawed sperm to identify factors causing cryo-injury. Ejaculates were collected (artificial vagina) from six Dorper rams, pooled, extended, and frozen over liquid nitrogen. Overall, 915, 3382, and 6875 phosphorylated proteins, phosphorylated peptides, and phosphorylation sites, respectively, were identified. At least two modified sites were present in 57.94% of the 6875 phosphosites identified, of which AKAP4 protein contained up to 331 modified sites. There were 732 phosphorylated peptides significantly up-regulated and 909 significantly down-regulated in frozen-thawed versus fresh sperm. Moreover, the conserved motif [RxxS] was significantly down-regulated in frozen-thawed sperm. Phosphorylation of sperm-specific proteins, e.g., AKAP3/4, CABYR, FSIP2, GSK3A/B, GPI, and ODF1/2 make them potential biomarkers to assess the quality of frozen-thawed ram sperm. Furthermore, these differentially phosphorylated proteins and modification sites were implicated in cryopreservation-induced changes in sperm energy production, fiber sheath composition, and various biological processes. We concluded that abnormal protein phosphorylation modifications are key regulators of reduced sperm motility. These novel findings implicated specific protein phosphorylation modifications in sperm cryo-injury. SIGNIFICANCE: This study used phosphorylated TMT quantitative proteomics to explore regulation of epigenetic modifications in frozen-thawed ram sperm. This experiment demonstrated that ram sperm freezing affects phosphorylation site modifications of proteins, especially those related to functions such as sperm motility and energy production. Furthermore, it is important to link functions of phosphorylated proteins with changes in sperm quality after freezing and thawing, and to clarify intrinsic reasons for sperm quality changes, which is of great importance for elucidating mechanisms of sperm freezing damage. Based on these protein markers and combined with cryoprotectant design theory, it provides a theoretical basis and data reference to study sperm cryoprotectants., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2023. Published by Elsevier B.V.)

Background: Examining semen cryopreservation in Calomys laucha offers valuable insights for reproductive research and species conservation., Objective: To determine the most effective sugar for the cryopreservation of C. laucha semen., Materials and Methods: Using 36 epididymides from C. laucha, semen samples were diluted in a 3% skimmed milk medium supplemented with one of four sugars (glucose, fructose, lactose, or sucrose) at a concentration of 0.3 M. These mixtures underwent a conditioning phase at 37 degree C for 10 min, cooled to -80 degree C for another 10 min, and were subsequently stored in liquid nitrogen., Results: Upon thawing, samples treated with lactose and glucose solutions show superior sperm motility, achieving 8.2% and 10.0% respectively, in contrast to the fructose (2.0%) and sucrose (4.1%) mixtures. Furthermore, samples preserved in glucose registered the highest sperm penetration rates, reaching 44.9%., Conclusion: Our findings suggest that a cryopreservation medium containing 0.3 M glucose can contribute to the safeguarding C. laucha rodent semen. https://doi.org/10.54680/fr24210110612.

During the freeze-thaw process, human spermatozoa are susceptible to oxidative stress, which may cause cryodamage and reduce sperm quality. As a novel mitochondria-targeted antioxidant, Mito-tempo has been used for sperm cryopreservation. However, it is currently unknown what role it will play in the process of sperm ultra-rapid freezing. The purpose of this study was to investigate whether Mito-tempo can improve sperm quality during ultra-rapid freezing. In this study, samples with the addition of Mito-tempo (0, 5, 10, 20, and 40 μM) to sperm freezing medium were selected to evaluate the changes in sperm quality, antioxidant capacity and ultrastructure after ultra-rapid freezing. After ultra-rapid freezing, the quality and antioxidant function of the spermatozoa were significantly reduced and the spermatozoa ultrastructure was destroyed. The addition of 10 μM Mito-tempo significantly increased post thaw sperm motility, viability, plasma membrane integrity and mitochondrial membrane potential (P < 0.05). Moreover, the DNA fragmentation index (DFI), ROS levels and MDA content were reduced, and the antioxidant enzyme (CAT and SOD) activities were enhanced in the 10 μM Mito-tempo group (P < 0.05). Moreover, Mito-tempo protected sperm ultrastructure from damage. In conclusion, Mito-tempo improved the quality and antioxidant function of sperm after ultra-rapid freezing while reducing freezing-induced ultrastructural damage., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

The parameters of sperm apoptosis and capacitation during liquid storage at 17°C can indicate the quality of pig sperm and the potential development of early embryos. However, the effect of kojic acid (KA) on semen preservation and its mechanism has not been fully understood. In this study, we discovered that adding KA to the diluent improved the antioxidant capacity of sperm mitochondria, maintained the normal structure of sperm mitochondria, and reduced sperm apoptosis. Western blot analysis revealed that KA prevented the release of Cytochrome c from mitochondria to the cytoplasm, reduced the expression of pro-apoptosis proteins cleaved Caspase-3 and cleaved Caspase-9, and increased the expression of the antiapoptosis protein Bcl-XL. Furthermore, KA also enhanced the motility parameters, oxidative phosphorylation level, adenosine triphosphate level, and protein tyrosine phosphorylation of capacitated sperm, while preserving the acrosome integrity and plasma membrane integrity of capacitated sperm. In conclusion, this study offers new insights into the molecular mechanism of how KA inhibits porcine sperm apoptosis and improves capacitated sperm parameters. Additionally, it suggests that KA can serve as an alternative to antibiotics., (© 2024 Wiley Periodicals LLC.)