Liquid-stored Semen Research Articles

Effect of selenium and vitamin E addition to the extender on liquid stored capercaillie (Tetrao urogallus) semen quality.

Captive breeding has become an important tool in species conservations programmes, maintaining genetic diversity and restoring wild, endangered populations. In order to improve the reproductive efficiency of captive kept capercaillie, the purpose of the study was to determine the effect of selenium and vitamin E addition to semen extender on sperm characteristic during short-term storage. Ejaculates collected individually from four capercaillie were divided into two parts, diluted threefold with basic EK extender and EK enriched with 1mg/ml of organic selenium and 8mg/ml of vitamin E (EK+Se+E) and stored 24hr at temp. +4°C. Spermatozoa morphology, motility and motility parameter were evaluated in net, diluted and stored semen samples. Significant (p<.05) differences between individual males were stated in relation to the majority of traits evaluated in the freshly collected semen. Comparing to the fresh semen, a significant (p<.05) decrease in percentage of live sperm in total (by 3.8% points on average) has been observed in samples diluted by EK extender, while in semen diluted with EK+Se+E extender this decrease was lower (1.5%pts on average) and not significant. Also per cent of motile sperm in EK+Se+E extender was higher (p<.05) then in EK (71.6% vs. 58.9%), but taking into account the values of individual males, both extender and male effect on liquid semen storage become apparent. Obtained data allow concluding that selenium and vitamin E addition to EK extender had positive effect on morphology and motility of capercaillie semen stored 24hr at 4°C and can be recommended for similar studies carried out on other Galliformes species.

Metabolic Activity of Boar Semen Stored in Different Extenders Supplemented with Ostrich Egg Yolk Lipoproteins.

IntroductionThe aim of this study was to evaluate the effect of lipoprotein fraction isolated from ostrich egg yolk (LPFo) on the metabolic activity of boar spermatozoa following liquid semen storage in different extenders and temperatures.Material and MethodsBoar ejaculates were extended in Androhep, Beltsville thawing solution (BTS), and Martín-Rillo and Alias (MR-A) without (control) or with the addition of LPFo and stored for three days at either 5°C or 16°C. The analysed sperm parameters included total motility (TMOT), plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), oxygen consumption, and adenosine triphosphate (ATP) production.ResultsThe sperm metabolic activity seemed to be higher in the LPFo-based extenders following storage for three days, irrespective of the storage temperature. Compared with the LPFo-free extenders, significantly higher (P < 0.05) sperm PMI and MMP were observed in BTS and MR-A extenders supplemented with LPFo during storage for three days at 5°C. Spermatozoa stored in the BTS-LPFo extender exhibited higher (P < 0.05) TMOT and oxygen consumption, whereas higher (P < 0.05) PMI was observed in spermatozoa stored in Androhep-LPFo and MR-A-LPFo for three days at 16°C. No significant differences (P > 0.05) in ATP content were observed between the LPFo-free and LPFo-based extenders during storage.ConclusionsSupplementation of LPFo to semen extenders had varying effects on the metabolic activity of boar spermatozoa stored at different temperatures. It can be suggested that the interactions of various components of the extenders and seminal plasma with LPFo exert beneficial effects on the sperm metabolic activity during liquid storage of boar semen.

State-of-the-art of boar sperm preservation in liquid and frozen state

Pig breeding is mainly conducted through Artificial Insemination (AI) in Western and developing countries. Apart from requiring specific catheters and trained staff, preserving boar semen in proper conditions is needed to ensure high reproductive performances. Although, at present, boar sperm may be preserved in liquid (15-17oC) or frozen states, more than 95% of AIs are conducted using liquid semen. The present work reviews the state-the-art of these two preservation technologies. Thus, the composition and types of extenders for liquid-stored semen are discussed, together with the specific requirements for boar sperm, which are stored at 15-17oC. Commercial extenders for liquid semen are compared and the effects of storage on sperm quality are also summarised. In the second part of the manuscript, the main features of boar sperm cryopreservation are described and reference to cryodamage is also made. These cryoinjuries mainly affect sperm motility, membrane permeability and chromatin integrity. Furthermore, the individual variability in the sperm resilience to withstand cryopreservation procedures is reviewed and a brief summary about freezability markers is also included. Final sections briefly discuss the improvement of freezing extenders with additives, such as seminal plasma and antioxidants, and highlight the relevance of using a proper AI technique to avoid dramatic drops in reproductive performance.

Glutathione Peroxidase 5 Is Expressed by the Entire Pig Male Genital Tract and Once in the Seminal Plasma Contributes to Sperm Survival and In Vivo Fertility.

Glutathione peroxidase-5 (GPX5) is an H2O2-scavenging enzyme identified in boar seminal plasma (SP). This study attempted to clarify its origin and role on sperm survival and fertility after artificial insemination (AI). GPX5 was expressed (Western blot and immunocytochemistry using a rabbit primary polyclonal antibody) in testes, epididymis and accessory sex glands (6 boars). SP-GPX5 concentration differed among boars (11 boars, P < 0.001), among ejaculates within boar (44 ejaculates, P < 0.001) and among portions within ejaculate (15 ejaculates). The first 10 mL of the sperm rich fraction (SRF, sperm-peak portion) had a significantly lower concentration (8.87 ± 0.78 ng/mL) than the rest of the SRF and the post-SRF (11.66 ± 0.79 and 12.37 ± 0.79 ng/mL, respectively, P < 0.005). Sperm motility of liquid-stored semen AI-doses (n = 44, at 15–17°C during 72h) declined faster in AI-doses with low concentrations of SP-GPX5 compared to those with high-levels. Boars (n = 11) with high SP-GPX5 showed higher farrowing rates and litter sizes than those with low SP-GPX5 (a total of 5,275 inseminated sows). In sum, GPX5 is widely expressed in the boar genital tract and its variable presence in SP shows a positive relationship with sperm quality and fertility outcomes of liquid-stored semen AI-doses.

Effects of glucose metabolism pathways on sperm motility and oxidative status during long-term liquid storage of goat semen

Although great efforts were made to prolong the fertility of liquid-stored semen, limited improvements have been achieved in different species. Although it is expected that energy supply and the redox potential will play an essential role in sperm function, there are few reports on the impact of specific energy substrates on spermatozoa during liquid semen storage. Furthermore, although it is accepted that glucose metabolism through glycolysis provides energy, roles of pentose phosphate pathway (PPP) and tricarboxylic acid cycle remain to be unequivocally found in spermatozoa. We have studied the pathways by which spermatozoa metabolize glucose during long-term liquid storage of goat semen. The results indicated that among the substrates tested, glucose and pyruvate were better than lactate in maintaining goat sperm motility. Although both glycolysis and PPP were essential, PPP was more important than glycolysis to maintain sperm motility. Pentose phosphate pathway reduced oxidative stress and provided glycolysis with more intermediate products such as fructose-6-phosphate. Pyruvate entered goat spermatozoa through monocarboxylate transporters and was oxidized by the tricarboxylic acid cycle and electron transfer to sustain sperm motility. Long-term liquid semen storage can be used as a good model to study sperm glucose metabolism. The data are important for an optimal control of sperm survival during semen handling and preservation not only in the goat but also in other species.

Dietary n-6:n-3 ratio and Vitamin E improve motility characteristics in association with membrane properties of boar spermatozoa.

This study was aimed to evaluate the effects of dietary n-6:n-3 ratio and Vitamin E on the membrane properties and motility characteristics of spermatozoa in boars. Forty Duroc boars were randomly distributed in a 2 × 2 factorial design with two n-6:n-3 ratios (14.4 and 6.6) and two Vitamin E levels (200 and 400 mg kg−1). During 16 weeks of treatment, fresh semen was collected at weeks 0, 8, 12, and 16 for measurements of motility characteristics, contents of fatty acids, membrane properties (membrane fluidity and membrane integrity), and lipid peroxidation of the spermatozoa. The semen was diluted in Beltsville Thawing Solution (BTS) extender and stored at 17°C, and the sperm motility was assessed at 12, 36, 72, and 120 h of storage. The 6.6 n-6:n-3 dietary ratio increased the contents of n-3 polyunsaturated fatty acids (PUFAs) and docosahexaenoic acid (DHA) and improved the membrane integrity and membrane fluidity of the spermatozoa, resulting in notably increased total motility, sperm progressive motility, and velocity parameters of fresh semen. Feeding diet with Vitamin E (400 mg kg−1) prevented sperm lipid peroxidation, and resulted in higher total motility and sperm progressive motility in fresh and liquid stored semen. In conclusion, the adjustment of n-6:n-3 ratio (6.6) and supply of Vitamin E (400 mg kg−1) successfully improved sperm motility characteristics and thus may be beneficial to the fertility of boars, which might be due to the modification of the physical and functional properties of spermatozoa membrane in response to dietary supplementation.

Effects of pH during liquid storage of goat semen on sperm viability and fertilizing potential

A specific problem in goat semen preservation is the detrimental effect of seminal plasma on sperm viability in extenders containing yolk or milk. Thus, the use of chemically defined extenders will have obvious advantages. Although previous studies indicate that the initial pH of an extender is crucial to sustain high sperm motility, changes in extender pH during long-term semen storage have not been observed. Monitoring extender pH at different times of semen storage and modeling its variation according to nonlinear models is thus important for protocol optimization for long-term liquid semen preservation. The present results showed that during long-term liquid storage of goat semen, both sperm motility and semen pH decreased gradually, and a strong correlation was observed between the two. Whereas increasing the initial extender pH from 6.04 to 6.25 or storage with stabilized pH improved, storage with artificially lowered pH impaired sperm motility. Extender renewal improved sperm motility by maintaining a stable pH. Sperm coating with chicken (Gallus gallus) egg yolk improved motility by increasing tolerance to pH decline. A new extender (n-mZAP) with a higher buffering capacity was formulated, and n-mZAP maintained higher sperm motility, membrane integrity and acrosome intactness than the currently used mZAP extender did. Goat semen liquid-stored for 12 d in n-mZAP produced pregnancy and kidding rates similar to those obtained with freshly collected semen following artificial insemination. In conclusion, maintenance of a stable pH during liquid semen storage dramatically improved sperm viability and fertilizing potential.

Motility of liquid stored ram spermatozoa is altered by dilution rate independent of seminal plasma concentration

The fertility after use of liquid stored ram semen following cervical AI rapidly decreases if semen is stored beyond 12h. The dilution of seminal plasma is often cited as a key contributor to the diminished motility and fertility of ram spermatozoa subjected to liquid preservation. Two experiments were conducted to assess the effect of spermatozoa concentration (i.e. dilution rate) and percentage of seminal plasma on the motility and viability of liquid stored ram spermatozoa. In Experiment 1, semen was diluted to one of seven concentrations ranging from 0.2 to 1.4×109spermatozoa/ml with milk and assessed for motility after 3 or 24h of storage at 15°C. In Experiment 2, semen was collected and washed to remove seminal plasma before re-dilution to 0.2–1.4×109spermatozoa/ml with milk containing 0%, 20% or 40% (final v/v ratio) seminal plasma and assessed for viability and motility after 3 or 24h of storage at 15°C. Whereas motility was not affected by spermatozoa concentration after 3h of storage, the proportion of progressive spermatozoa decreased after 24h of storage when spermatozoa concentration was greater than 1.0×109spermatozoa/ml. The duration of preservation and the spermatozoa concentration affected spermatozoa motility but had no impact on spermatozoa viability. This negative effect of greater spermatozoa concentrations on motility was independent of the presence and the concentration of seminal plasma. The seminal plasma at both concentrations (20% and 40%) had a protective effect on spermatozoa motility after 24h of storage. These findings have the potential to improve the efficiency of cervical AI with liquid stored ram semen.

The activity of paraoxonase type 1 (PON-1) in boar seminal plasma and its relationship with sperm quality, functionality, and in vivo fertility.

Paraoxonase 1 (PON-1) is a hydrolytic enzyme present in body fluids, capable of protecting cells against oxidative stress. The hypothesis was hereby to test that PON-1, present in seminal plasma (SP), acts protecting boar spermatozoa when showing a reasonable high activity in the ejaculate. SP-PON-1 activity differed (p < 0.001) among boars (from 0.10 to 0.29 IU/mL). Intra-boar variability was also observed (p < 0.05), but only in two of the 15 boars. SP-PON-1 activity differed among ejaculate portions, showing the spermatozoa-peak portion of spermatozoa-rich ejaculate fraction the highest levels (0.35 ± 0.03 IU/mL, ranging from 0.12 to 0.69) and the post-sperm ejaculate fraction the lowest levels (0.12 ± 0.01 IU/mL, ranging from 0.03 to 0.21). SP-PON-1 activity was positively correlated with the percentage of spermatozoa with rapid and progressive movement (p < 0.01) and negatively correlated with the generation of intracellular reactive oxygen species (p < 0.01) in semen samples after 72 h of liquid storage. SP-PON-1 activity was highest (p < 0.01) in boars with highest farrowing rates. In conclusion, SP-PON-1 activity differed among boars and ejaculate fractions/portions. SP-PON-1 activity was positively correlated with sperm quality and functionality of liquid-stored semen samples and it evidenced a positive association with in vivo fertility.

Highlights on Artificial Insemination (AI) Technology in the Pigs

Abstract Over the past decade, there has been a tremendous increase in the development of field AI services in the majority of countries concerned with pig production. The objective of this paper is to review: (a) the current status of swine AI in the world, (b) significance and limitation of AI with liquid and frozen semen, (c) the biological traits of porcine semen in relation to in vitro sperm storage, (d) the criteria used for selection of a boar stud as a semen supplier, (e) how to process boar semen for liquid and frozen storage in the commercial settings and (f) how to improve fertility and prolificacy of boar semen. More than 99% of the inseminations conducted worldwide are made with liquid-stored semen. AI with frozen semen is used only for upgrading the genetic base in a particular country or herd. Determining the initial quality of semen ejaculates along with the selection of the optimum storage extender has a profound effect on the quality and fertility of AI doses. Different procedures have been used for improving the fertility of preserved spermatozoa including colloidal centrifugation of the semen, intrauterine insemination and modulation of the uterine defense mechanism after AI. Development of an efficient protocol for synchronizing the time of ovulation in sows and gilts coupled with improving uterine horn insemination technique will make a breakthrough in the commercial use of frozen boar semen.

The effect of dietary n-3 polyunsaturated fatty acids supplementation of rams on semen quality and subsequent quality of liquid stored semen

The objective of this study was to examine the effect of dietary n-3 polyunsaturated fatty acid (PUFA) supplementation of rams on semen quality and subsequent sperm function of liquid stored semen. Mature rams of proven fertility were individually housed and were blocked according to breed, body weight, and body condition score and randomly allocated within block to one of two dietary treatments (N = 7 per treatment). Rams were offered a base diet of hay and concentrate, with the concentrate enriched with either: (1) saturated palmitic acid (CON) or (2) high n-3 PUFA fish oil (FO) supplements. Both lipid supplements were added at 2% (wt/wt) of the total diet as fed and both were partially rumen-protected. The animals were fed their respective diets for a total of 9 weeks and blood samples were collected on weeks 0 (pre-experimental), 4, and 9, relative to initial allocation of diet (week 0), for measurement of plasma concentration of fatty acids, metabolites, insulin like growth factor 1 (IGF-1) and insulin. Semen was collected from each ram (on 1 day in each week) in weeks 4, 5, 7, 8, and 9, and each ejaculate was assessed for volume, wave motion, and concentration of sperm, after which it was diluted in a skim milk-based extender and stored at 4 °C. A second ejaculate was collected on weeks 4, 7, and 9, centrifuged, and the sperm frozen for subsequent lipid analysis. A sample of semen from each ram was assessed at 24, 48, and 72 hours after collection for sperm progressive linear motion, ability to penetrate artificial mucus, and the ability to resist lipid peroxidation (at 24 and 48 hours only) using the thiobarbituric acid reactive substances assay. There was no effect of diet on plasma insulin concentrations or on any of the metabolites measured, however, there was a diet by week interaction for plasma IGF-1 concentration (P < 0.05). This was manifested as the FO supplemented rams having higher IGF-1 concentrations on week 9 compared with the control treatment (P < 0.05), but not at the earlier sampling dates. Compared with the pre-experimental values, supplementation with FO increased plasma concentrations of total n-3 PUFAs by 3.1-fold and decreased n-6 PUFA concentrations by 1.84-fold. Consequently, the ratio of n-6 to n-3 PUFA was decreased in the FO-supplemented rams (P < 0.001). Dietary supplementation with FO increased the concentration of eicosapentaenoic acid in sperm from week 4 to 9 by 2.7-fold (P < 0.05) leading to a 1.5-fold increase in total n-3 PUFA in the same period. Ejaculates collected from rams supplemented with FO yielded a higher semen concentration (P < 0.05), however, there was no difference between diets on any of the other semen quality parameters including semen volume, wave motion, progressive linear motion, ability to penetrate artificial mucus, or ability to resist lipid peroxidation. In conclusion, dietary supplementation of rams with n-3 PUFA successfully increased the n-3 PUFA content of plasma and sperm but has limited effects on the quality of liquid stored semen.

The effect of bovine serum albumin and fetal calf serum on sperm quality, DNA fragmentation and lipid peroxidation of the liquid stored rabbit semen

The aim of the present study was to determine the effects of the bovine serum albumin (BSA) and fetal calf serum (FCS) on sperm quality, DNA fragmentation and lipid peroxidation of liquid stored rabbit semen stored up to 72h at 5°C. Ejaculates were collected from five New Zealand male rabbits by artificial vagina and pooled at 37°C following evaluation. Each pooled ejaculate was split into three equal experimental groups and diluted to a final concentration of approximately 40×106sperm/ml (single step dilution), in an Eppendorf tube, with the Tris based extender containing BSA (5mg/ml), FCS (10%) or no additive (control) at 37°C, cooled to 5°C and stored for up to 72h. The extender supplemented with BSA and FCS did not improve the percentages of motility and acrosomal abnormality during 48h compared to the control. The additives BSA and FCS had a significant effect in the maintaining of plasma membrane integrity between 48 and 72h storage period, compared to the control (P<0.01). The supplementation of BSA and FCS had a protective effect on motility (P<0.05), plasma membrane integrity (P<0.01) and acrosomal integrity (P<0.01) at 72h compared to the control. The supplementations with BSA and FCS led to a reduction in DNA damage of rabbit sperm at 48 and 72h during storage period, compared to the control (P<0.001). Although supplementation of BSA and FCS caused significant (P<0.01) decreases in malondialdehyde (MDA) level at 48h and 72h, they significantly (P<0.01) increased the glutathione peroxidase (GPx) antioxidant activity up to 72h when compared to the control group. In conclusion, BSA and FCS supplementation to liquid stored rabbit semen provide a protection for spermatozoa against cool storage-induced DNA damage and plasma membrane integrity by their antioxidative properties.

Study on the Ejaculate Characteristics and Liquid Storage of Semen in the Common Bottlenose Dolphin (Tursiops truncatus)

Study on the Ejaculate Characteristics and Liquid Storage of Semen in the Common Bottlenose Dolphin (Tursiops truncatus)

Farklı Saklama Isılarının Kısa Süreli Saklanan Yeni Zelanda Tavşan Spermatozoasının DNA Hasarı ve Sperm Parametreleri Üzerine Etkisi

Summary The effect of two different temperatures (4 o C and 15 o C) on motility, plasma membrane integrity, acrosome abnormality and DNA damage of rabbit spermatozoa was evaluated at 0 and 24 h of liquid storage. Ejaculates collected from six New Zealand male rabbits by artificial vagina and pooled at 37 o C following evaluation. Pooled ejaculate was divided into two equal aliquots and diluted with the Tris based semen extender at a final concentration of approximately 40x10 6 sperms/ml in a Eppendorf plastic tube. There were no significant differences in the percentage of above mentioned parameters between 4 o C or 15 o C at the beginning of liquid storage (0 h). The percentages of motility (75.0±1.83%) and plasma membrane functional integrity (71.2±1.14%) at 15 o C was significantly better than that of liquid stored semen at 4 o C (67.9±1.01% and 65.3±1.38%, P<0.05, respectively) at 24 h of storage. The percentage of acrosome abnormality at 24 h wasn’t affected by the different storage temperature. The influence of storage temperature and the length of time on spermatozoa DNA damage was found statistically significant (P<0.001). The storage period for up to 24 h lead to an increase in the percentage of spermatozoa DNA damage (P<0.001). The percentages of DNA damage at 4 o C was statistically higher than 15 o C (P<0.001). In conclusion, 15 o C may be prefered when liquid stored rabbit semen are used for 24 h.

Age-related changes in quality and fertility of porcine semen

The aim of this study was to investigate the effect of boar age on quality traits and fertility of liquid-stored semen. Boars were allocated into 3 age groups: 7-10 months (young), 18-33 months (mature), 51-61 months (old). Ejaculates of > 200x10(6) sperm/ml and 85% total motile sperm were extended to 30x10(6) sperm/ml, stored at 17-18 °C and used within 12-24 h for artificial insemination (AI) of 2062 multiparous sows. After 24 h of storage, aliquots of diluted semen were assessed for sperm progressive motility (SPM), incidence of sperm chromatin instability (SCI), proportion of live morphologically normal sperm (LMNS) and head morphometry of LMNS. The results showed that young boars had higher percentages of SCI and lower proportions of LMNS than those of the mature (p < 0.05) and old (p < 0.001) boars, respectively. Sperm head dimensions of young and old boars were greater (p < 0.03-0.001) than those of mature boars. The farrowing rate of young boars (65%) was significantly lower (p < 0.001; χ2= 30-61) than those of the mature (87.2%) and old (84.7%) boars. The relationship between sperm head dimensions and boar fertility was non-significant. In conclusion, boar age is an important physiological factor contributing to the success of swine AI.

Successful preservation of capercaillie (Tetrao urogallus L.) semen in liquid and frozen states

Experiments on semen collection and preservation were undertaken by Wrocław University of Environmental and Life Sciences and Forestry Wisła, Poland to assist in the protection of the capercaillie (Tetrao urogallus L.) and to create an ex situ in vitro cryobank. Semen was collected from 11 captive-bred males, using dorsoabdominal massage. Ejaculates once obtained were diluted 3-fold at room temperature with EK diluent and then a number of them were stored at 4 °C for 18, 24, and 48 hours, while the remaining ejaculates were equilibrated with 6% dimethylacetamide and frozen by pipetting, drop-by-drop directly onto a liquid nitrogen surface. Frozen pellets were thawed at 60 °C in a water bath after 4 to 28 mo of storage. In total, 103 individually collected ejaculates (54 stored as liquid and 49 frozen in liquid nitrogen) were of appropriate value for further processing. The volume of ejaculates varied from 30 to 240 μL; spermatozoa concentration from 70 × 106 mL−1 to 1950 × 106 mL−1. The total amount of live spermatozoa in the fresh semen varied from 85.3% to 99.0%, of which from 41.1% to 85.3% were morphologically normal. Among morphologically abnormal forms, bulb-head (5.6% to 36.0%) and midpiece deformations (1.3% to 16.6%) were the most frequent. Dilution and semen storage up to 24 h at 4 °C did not affect the semen quality, as far as motility and sperm morphology are concerned. A significant (P < 0.05) decrease in total live (94.9 vs. 91.7%) and live normal cells (66.4 vs. 56.7%) was observed after 48 h. About 30% to 40% of spermatozoa remained motile. Cryopreservation significantly decreased (P < 0.05) the total number of live and live normal spermatozoa however, in relation to the fresh semen, their average content was 44.1% and 37.4%, respectively. Significant (P < 0.05) individual differences were observed in the quality of the fresh, liquid stored and the frozen-thawed semen assessed in terms of spermatozoa motility and morphology. After a single insemination with thawed semen containing 9.7 million live normal cells, 80% fertility and 100% hatchability were achieved. The obtained results indicate for the first time that there is the potential to use liquid stored and cryopreserved capercaillie semen to support conservation measures for the maintenance of genetic diversity, as well as to increase the number of reintroduced progeny of this endangered grouse species.

Boar variability affects sperm metabolism activity in liquid stored semen at 5°C

Metabolic activity of boar spermatozoa, liquid stored for three days at 5 degrees C, was measured using bioluminescence for ATP content, fluorescent assay (JC fluorochrome) of mitochondrial activity and oxygen consumption. Sperm motility and plasma membrane integrity (PMI) were simultaneously analyzed. Apart from the statistically significant effect (P < 0.001) of semen storage time, the importance of the individual source of the ejaculate for the analyzed parameters of metabolic efficiency of spermatozoa was shown. This phenomenon was manifested in the interaction of the individual source of the ejaculate with spermatozoa motility, integrity of their membranes and metabolic activity with the passing time of semen preservation. Recorded results indicate that the individual factor may have a significant influence on the technological usefulness of boar spermatozoa for liquid storage. Quality analyses conducted on boar semen stored at 5 degrees C may be used for pre-selection of boars producing sperm with an enhanced tolerance to cold shock.

Liquid Semen Storage in Elephants (Elephas maximus and Loxodonta africana): Species Differences and Storage Optimization

Artificial insemination plays a key role in the genetic management of elephants in zoos. Because freshly extended semen is typically used for artificial insemination in elephants, it has become imperative to optimize conditions for liquid storage and semen transport. The objectives of this study were to examine the interactions between different extenders and storage temperatures on sperm total motility, progressive motility, and acrosomal integrity in Asian (Elephas maximus) and African (Loxodonta africana) elephants. Ejaculates were collected by rectal massage, diluted using a split-sample technique in 5 semen extenders: TL-Hepes (HEP), Modena (MOD), Biladyl (BIL), TEST refrigeration medium (TES), and INRA96 (INR), maintained at 35°C, 22°C, or 4°C. At 0, 4, 6, 12, and 24 hours, aliquots were removed and assessed for sperm total motility, progressive motility, and acrosomal integrity. After 24 hours of storage, African elephant spermatozoa exhibited greater longevity and higher values in sperm quality parameters compared with those of Asian elephants. In both species, semen storage at 35°C resulted in a sharp decline in all sperm quality parameters after 4 hours of storage, whereas storage at 22°C and 4°C facilitated sperm survival. In Asian elephants, MOD and HEP were most detrimental, whereas BIL, TES, and INR maintained motility up to 12 hours when spermatozoa were cooled to 22°Cor4°C. In African elephants, there were no differences among extenders. All media maintained good sperm quality parameters at 22°C or 4°C. However, although MOD, BIL, and INR were most effective at lower temperatures, HEP and TES maintained sperm motility at all storage temperatures. This study demonstrated sperm sensitivity to components of various semen extenders and storage temperatures and offers recommendations for semen extender choices for liquid semen storage for both Asian and African elephants.

Influence of methionine and dithioerythritol on sperm motility, lipid peroxidation and antioxidant capacities during liquid storage of ram semen

The aim of this study was to investigate the effects of methionine and dithioerythritol, added to the Tris extender, on ram sperm motility and LPO (lipid peroxidation) and antioxidant capacities during liquid storage up to 72 h at 5 °C. Ejaculates collected from five Merino rams, were evaluated and pooled at 37 °C. This study included two experiments. In experiment 1, each pooled ejaculate was divided into four equal aliquots and diluted (37 °C) with the base extender, containing 0 (control), 1, 2 and 4 mM methionine, at a final concentration of approximately 4 × 10 8 sperms/ml (single step dilution), in a 15-ml plastic centrifuge tube. In experiment 2, dithioerythritol, at concentrations of 0 (control), 0.5, 1 and 2 mM, was used as an additive in the extender, and the procedure explained above was applied for the division of aliquots and the dilution of semen. Diluted semen samples were kept in glass tubes and cooled from 37 to 5 °C in a cold cabinet, and maintained at 5 °C. Sperm motility and LPO and total glutathione (GSH) and glutathione peroxidase (GPx) capacities were determined at 5 °C for periods of 0, 24, 48 and 72 h of liquid storage. The extender supplemented with 1 mM methionine led to higher motility percentages (77.0 ± 1.2%), in comparison to the control group (66.0 ± 4.9%), during 72 h of liquid storage ( P < 0.05). As regards dithioerythritol, it did not statistically improve the motility rates for any of the storage times at 5 °C. In biochemical assays, differences in LPO levels between the groups with antioxidants and the control groups were not statistically significant. Compared to the control group, no significant difference was observed in GSH and GPx activities following the addition of methionine, during 72 h of storage. Total GSH and GPx activities did not increase significantly upon supplementation with 0.5 and 1 mM of dithioerythritol, compared to the control group, at any of the time points ( P > 0.05). Dithioerythritol at 2 mM led ( P < 0.01) to elevating GSH activity, compared to the control group, during 72 h of liquid storage. GPx activity was approximately 10 times higher for 2 mM of dithioerythritol ( P < 0.001), compared to that of the control group at all time points. The question regarding the sustainability of sperm survival, LPO and antioxidant capacities following liquid storage of semen remains unanswered. Further studies are required for a better understanding of the biochemical changes and to obtain more information on the determination of lipid peroxidation and antioxidant capacities during cooled storage of ram semen.

Effect of different storage temperatures on the metabolic activity of spermatozoa following liquid storage of boar semen

This study investigated the effects of different temperatures (5o and 16oC) on the metabolic activity of boar spermatozoa preserved in a standard semen extender, Kortowo 3 (K3), supplemented with or without egg yolk lipoprotein fractions (LPF) isolated from hen or ostrich egg yolk. Besides motility and plasma membrane integrity (PMI) assessments of spermatozoa, analysis of the metabolic activity of spermatozoa included mitochondrial energy status, oxygen uptake, ATP content and L-lactate production. Total motility and PMI of spermatozoa decreased over time. The metabolic activity of spermatozoa was significantly higher in the extenders containing LPF than in the K3 extender. Marked changes in the sperm metabolic activity observed during storage of semen samples at 5oC indicated that glycolysis (fructolysis) was the major metabolic pathway. It seemed likely that mitochondrial respiration was the predominant metabolic activity exhibited by spermatozoa during storage at 16oC. The findings of this study indicate that different storage temperatures can modulate the metabolic pathways of boar spermatozoa during liquid semen storage.