Liquid Semen Research Articles

Overview of Turkey Semen Storage: Focus on Cryopreservation – A Review

Abstract This review updates the current state of technologies available for turkey semen storage (hypothermic- liquid storage and cryopreservation), with special attention paid to cryopreservation. Liquid semen can be stored for up to 24 or 48 h at temperatures around 5°C, while cryopreservation allows long-term storage at -196°C. The possibility of using frozen turkey semen for artificial insemination (AI) would have practical benefits for turkey production. Reported fertility rates in response to AI using frozen/thawed semen range from 15.8 to 84.3%. Unsatisfactory fertility may be attributed to an inability of turkey spermatozoa to successfully survive the freezing/thawing process, and this, along with the high variability observed, makes this technique unacceptable for commercial breeding programs. There is therefore a need to standardize the whole freezing and thawing process to improve the post-thaw quality of turkey semen and minimize variability in results. Finding an efficient freezing protocol for turkey semen will allow for the creation of a sperm cryobank, improving current prospects for the commercial use of frozen turkey semen and also for the long-term conservation of the genetic diversity of this bird.

Association study analysis of phospholipase C zeta gene polymorphism forsperm motility and kinematic characteristics in liquid semen of Boar

Association study analysis of phospholipase C zeta gene polymorphism forsperm motility and kinematic characteristics in liquid semen of Boar

PENENTUAN KONSENTRASI SODIUM DODECYL SULFATE DALAM PENGENCER RINGER LAKTAT-KUNING TELUR UNTUK PRESERVASI SEMEN AYAM PELUNG (Determination of Sodium Dodecyl Sulfate Concentration in Ringer Lactate-Egg Yolk Extender for Pelung Rooster Semen Preservation)

The objective of this experiment was to determine the addition of sodium dodecyl sulfate (SDS) to Ringer Lactate-Egg Yolk (RL-EY) extender on pelung chicken semen preservation. Semen was collected three times a week from three pelung chickens. Collected semen was evaluated macroscopically and microscopically. Only ejaculates of at least 70% sperm motility was then divided into three equal microtubes. Each of them diluted with RL-EY with ratio 90%:10%, then added with 0.00, 0.025, and 0.05% SDS respectively. The liquid semen then stored at 5 °C for 72 hours. Sperm motility and viability were observed every 12 hours. The addition of 0.025% SDS showed higher spermatozoa motility and viability (72.08±1.44% and 80.82±1.30%) which were significantly higher (P<0.05) than 0.00 and 0.05% SDS addition at 24 hours of storage. There was no differences on the spermatozoa motility and viability between 0.00 and 0.05% SDS addition. The decrease of spermatozoa motility and viability was observed in 0.025% SDS addition (4.17±0.56% and 4.65±0.59%, respectively) that significantly lower compared to 0% and 0.05% SDS addition at 24 hours of storage. In conclusion, 0.025% SDS addition in a Ringer Lactate -Egg Yolk extender maintained pelung spermatozoa motility and viability better than 0.00 and 5% SDS addition.

Effect of breeding season on fertility of sheep following estrus synchronization and fixed-time artificial insemination under field conditions in semi-arid tropical region

Accelerated lambing system is heavily reliant on reproductive technologies meant to enable off the season breeding in sheep. Therefore, the present study was programmed to assess the effect of breeding season (BS) on fertility of sheep following estrus synchronization (ES) and fixed time artificial insemination (FTAI). A total of 248 and 365 ewes were synchronized and inseminated during the BS (Febuary–March and July–September) and non-breeding season (NBS) respectively, during 2012–14. Synchronization of estrus was done by AVIKESIL-S, intra-vaginal progesterone sponges kept in situ in vagina for 12 days. 200 IU eCG was administered intramuscularly on 12th day after sponge withdrawal. FTAI was performed twice in ewes exhibiting signs of estrus at 48 and 56h after sponge removal, using liquid chilled semen of Patanwadi/Malpura rams containing 100 million sperm. Breeding season had no significant (p<0.05) effect on estrus synchronization. The estrous responses during the BS and NBS were 84.68% and 83.29% respectively. The lambing percentage during BS was 66.67%, which is significantly (p<0.05) higher than the lambing percentage of NBS (57.57%). Although, the lambing percentage in NBS maneuvered ewes were lower than the BS ewes but still the technology can be used to offset the effect of anestrus and to augment production in sheep.

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.

Pengaruh pengencer Tris Aminomethane kuning telur yang disuplementasi sari kulit Manggis (Garcinia Mangostana) terhadap kualitas semen Sapi Limousin selama penyimpanan suhu dingin 50C

The purpose of this research was to determine the dose of mangosteen rinds juice (0%, 2%, and 4%) in a diluent Tris Aminomethane -egg yolk. The fresh semen was collected once a week using an artificial vagina from five Limousin bulls aged 8 to 11 years old with a body weight of 895-1034 kg. The research method was experimental laboratory using a randomized block design with 3 treatments and 10 replications. If there were significant influences among the variables, it would be then tested by Duncan’s Multiple Range test method. The result showed that Tris Aminomethane-egg yolk diluent with 4% of mangosteen rinds juice suplementation had significant differences (P<0.05) on the sperm quality kept for 72 hours in the cold storage in terms of the percentage of sperm motility and sperm viability. It also had a significant difference (P<0.05) on sperm abnormality particularly within the 1 st hour. It can be concluded that the addition of 4% mangosteen rinds juice in Tris Aminomethane-egg yolk diluent could protect Limousin bull semen quality (motility, viability and abnormality) after stored for 72 hours at 5 0 C. Keywords : Abnormality, liquid semen, mangosteen rind, motility, viability

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.

The effect of extender, method of thawing, and duration of storage on in vitro fertility measures of frozen–thawed boar sperm

Frozen–thawed boar sperm (FTS) has reduced in vitro and in vivo life span compared to liquid semen. Experiments tested whether extenders, thawing procedures, and storage temperatures could extend the fertile life span of FTS. Experiment 1 tested the effect of six extenders on postthaw motility (MOT) and viability (VIA). Straws from boars (n = 6) were thawed, diluted into each extender, and evaluated at 20, 60, and 120 minutes. There was a trend (P = 0.08) for an extender-by-time interaction for MOT and effect of extender and time for MOT (P < 0.0001) and extender (P = 0.10) and time (P < 0.0001) for VIA. Experiment 2 evaluated the effect of temperature and time of thawing on in vitro fertility at intervals after thawing. Straws (0.5 mL) from different boar ejaculates (n = 15) were thawed at 50 °C for 10, 20, or 30 seconds or at 70 °C for 5, 10, or 20 seconds and evaluated at 5, 30, and 60 minutes. There was an effect of thawing treatment on MOT, VIA, and ACR (viable sperm with intact acrosomes, P < 0.0001) and an effect of time of evaluation (P < 0.0001) on MOT and ACR. Thawing at 70 °C for 20 seconds reduced (P < 0.05) MOT, VIA, and ACR compared to other treatments. Experiment 3 tested the effects of storage temperature and time after thawing using 20 ejaculates. Samples were thawed, diluted, and allotted to storage at 17 °C, 26 °C, or 37 °C with evaluation at 2, 6, 12, and 24 hours. There was a storage temperature and time effect and an interaction for MOT and VIA (P < 0.0001). Storage at 17 °C and 26 °C increased (P < 0.05) MOT over all times (38.5%) compared to 37 °C (26%), whereas MOT was reduced at intervals. Viability was also greatest with 17 °C and 26 °C compared to 37 °C and was also affected by time and decreased with time. These results indicate that FTS can be held at 17 °C or 26 °C for up to 2 hours before use and would allow for preparation of multiple doses. These data suggest in vitro fertility of FTS is affected by extenders, thawing, and storage.

The impact of storage temperature and sperm number on the fertility of liquid-stored bull semen.

In Ireland, liquid bull semen is stored at unregulated ambient temperatures, typically at 5×106 spermatozoa per dose, and inseminated within 2.5 days of collection. In Experiment 1, the effect of storage temperature (5, 15, 22, 32°C and fluctuations (Flux) between these temperatures) on progressive motility, viability, acrosomal status, DNA fragmentation and osmotic resistance was assessed. In Experiment 2, the field fertility of liquid semen at 5, 4 and 3×106 spermatozoa per dose, up to Day 2 after collection, was assessed in comparison to frozen-thawed semen at 20×106 spermatozoa per dose (n=35328 inseminations). In Experiment 1, storage at 15°C resulted in the highest progressive motility (PP6 spermatozoa per dose on Day 2 of storage was reduced in comparison to frozen-thawed semen (P<0.01). In conclusion, liquid semen is versatile between storage temperatures of 5 and 22°C, but demonstrates reduced fertility on Day 2 of storage at lower sperm numbers in comparison to frozen-thawed semen.

Effect of CIDR on Conception Rate in Repeat Breeding Cattle Inseminated with Liquid Semen

A trial was conducted to study the effect of CIDR on conception rate (CR) in repeat breeding (RB) Vrindavani cows following insemination with liquid semen. After insemination with liquid semen, the experimental RB cows were divided into two groups: group-I (n=13) was treated with CIDR between day 5 and 18 post-insemination, while group-II (n=12) was kept as negative control. Pregnancy diagnosis was done on day 60 post-insemination by per rectal palpation. Conception rate (%) in CIDR treated group was 5/13 (38.46%), whereas, it was 2/12 (16.67%) in the control group. Though the increase of CR by 21.79% in the CIDR treated group was statistically non-significant as compared to the control.

The effect of numbers of frozen-thawed boar sperm and addition of prostaglandin F2α at insemination on fertility in pigs

Frozen-thawed boar sperm (FTS) has reduced fertility compared to liquid semen. Exogenous prostaglandin administered at insemination has been reported to improve cases of low fertility. This experiment tested the effect of number of FTS and addition of prostaglandin (PGF2α) on fertility. The experiment was performed in replicates using weaned sows (n=24) and synchronized gilts (n=94). All females were induced into estrus using PG600® at weaning or following estrus synchronization. At estrus, females received 0.5, 1.0, or 2 billion motile FTS (n=9 boars) with 0 or 5mg of PGF2α added into each AI dose at insemination. Inseminations occurred at 24 and 36h after onset of estrus and ovulation was monitored by ultrasound. Pregnancy and litter size were determined for sows at farrowing and d 50 of gestation for gilts at slaughter. There was no effect of PGF2α and no interaction with dose of FTS or parity on fertility (P>0.10). Pregnancy rate was affected by FTS dose (P<0.001) with 2.0×109 (76.3%) greater than 0.5×109 (46.2%) and 1.0×109 sperm (48.8±8.0%). Pregnancy rate was not affected by parity (P>0.10) but was influenced by boar (P<0.05). Number of fetuses was also affected by FTS dose (P<0.001) with 2.0×109 (10.1) and 1.0×109 (9.4) producing more pigs than 0.5×109 sperm (6.9±0.9). Litter size was also affected by parity (P=0.001) and boar (P<0.01). These results indicate that AI using 2.0×109 FTS can result in acceptable pregnancy rates and litter sizes but with no measurable benefit for addition of prostaglandin.

Application of liquid semen technology improves conception rate of sex-sorted semen in lactating dairy cows

The objective was to compare reproductive performance of liquid sex-sorted (SS) semen with that of conventional (CON) semen in lactating dairy cows. Between 2011 and 2013, commercial dairy herds (n=101, 203, and 253 for 2011, 2012, and 2013, respectively) with predominantly Holstein-Friesian cows were enrolled in a contract mating program to produce surplus heifers for export using liquid SS semen. During the spring mating period, each herd was allocated with liquid SS semen at 50% of its daily requirement and the remaining daily requirement was allocated with CON liquid semen. Sperm for producing SS semen was sorted by Sexing Technologies NZ Ltd. (Hamilton, New Zealand) and then packaged using the liquid semen technology of LIC (Hamilton, New Zealand) at a dose of 1×106 sperm. Artificial insemination (AI) with liquid SS semen was carried out between 43 and 46h after collection. Conventional semen straws contained 1.25×106, 1.75×106, or 2×106 sperm for semen to be used on d 1, 2, or 3 after collection, respectively. Only CON inseminations on the same days as when SS semen was used were included in the comparison. Herd managers biased usage of SS semen toward cows with a longer postpartum interval before the mating start date (64.0 vs. 62.8 d), cows of higher genetic merit (NZ$107.0 vs. NZ$98.4), younger cows (5.1 vs. 5.2yr), and cows in which they had more confidence of being genuinely in estrus as measured by a lower percentage of short returns between 1 and 17 d (5.3 vs. 7.5%). After adjusting for these factors, the estimated difference in nonreturn rate between AI with SS and CON semen over the 3 seasons was −3.8 percentage points (SS=70.2% vs. CON=74.0%; SS/CON=94.9%). The estimated maximum difference in calving rate per AI between SS and CON semen was −3.1 percentage points for 2011 (SS=51.2% vs. CON=54.3%; SS/CON=94.3%) and −3.0 percentage points for 2012 (SS=49.7% vs. CON=52.6%; SS/CON=94.5%). Calving data for 2013 were not yet available. The percentage of heifer calves born to AI with SS semen was 87.0% for 2011 and 85.8% for 2012, both of which were lower than the expectation of 90% mainly due to misidentification of calf dams in seasonal dairy herds calving on pasture. In summary, results in this report showed that liquid SS semen only required half the dose rate of frozen SS semen to achieve a reproductive performance of over 94% of CON semen in lactating dairy cows. Careful planning and a robust distribution network are required to avoid semen wastage and to maximize the benefit of liquid SS semen.

The use of oxytocin in liquid semen doses to reduce seasonal fluctuations in the reproductive performance of sows and improve litter parameters—a 2-year study

The objective of the present research was to eliminate seasonal fluctuations in year-round reproductive performance of sows and to improve litter parameters by administration of oxytocin into liquid semen insemination doses. A 2-year experiment was performed on crossbreed sows, Polish Large White × Polish Landrace, which were partitioned into two groups: control, insemination without any modification with 100 mL semen doses and oxytocin, insemination with 100 mL semen doses to which 5 IU of oxytocin was added just before insemination. A total of 10,486 inseminations were made. The farrowing rate and obtained litter parameters, including the effect of season, were analyzed. For each litter, the following factors were defined: average litter size, percentage of fetal death and mummified piglets, average piglet birth weight, percentage of piglet mortality, fecundity index, average number of piglets weaned, weaned piglet weight, and daily gain. Sows presented a positive reaction to the experimental factor. A statistically higher farrowing rate for oxytocin group in summer and autumn seasons was confirmed (P ≤ 0.01). Regardless of the season, a higher average litter size was observed in the oxytocin group with the most evident differences for winter, spring (P ≤ 0.01), and summer (P ≤ 0.05). The effect of oxytocin on the percentage of fetal death and mummified piglets born was not confirmed statistically except for winter. Analyzing the fecundity index, higher values were obtained for the oxytocin group in all seasons (P ≤ 0.01), including the lowest difference between groups for winter (51.43) and the highest for summer (100.61). A higher average birth piglet weight and weaned piglet weight were recorded for the oxytocin group in all seasons. The highest differences in birth piglet weight between groups were noted for spring (0.22 kg; P ≤ 0.01) and winter (0.17 kg; P ≤ 0.05) and in weaned piglet weight for winter and spring (0.58 kg and 0.52 kg; for both, P ≤ 0.01). The greatest daily gains were observed in the winter season (P ≤ 0.05) in favor of oxytocin. On the basis of the presented results, it should be noted that the use of oxytocin into insemination doses improves the farrowing rate and other parameters of the reproductive performance of sows. In the absence of negative effects, year-round insemination with oxytocin addition into seminal doses is recommended, which effectively improves the production performance and reduces the problem of seasonality in reproduction.

Efficacy of different extenders in preservation of liquid hampshire boar semen at 15°c

The present study was designed to compare the efficacy of different liquid semen extenders for preservation of boar semen at 15°C. Twenty ejaculates, four from each of five Hampshire boars were used to study the efficacy of BTS, GEPS, FEY and LSEEY extenders for preservation of boar semen at 15°C up to 72 h. The mean sperm motility, live sperm and live spermatozoa with intact acrosome were found to be significantly (P < 0.05) higher in GEPS as compared to other extenders. The sperm motility, live sperm and live spermatozoa with intact acrosome differed significantly (P < 0.01) between extenders, preservation periods and interaction of extenders and preservation periods. GEPS was found to be the best extender for preservation followed by BTS, LSEEY and FEY.

Preservation Effect of Crude Fig Fruit Filtrate (Ficus carica L) Added In to Tris Egg Yolk Based Extender on Capacitating, Acrosome and Fertility of Half Blood Boer Buck Spermatozoa

This research was conducted to know the effect of additional crude filtratet of fig fruit (CF3) into tris egg yolk (TEY) based extender on sperm progressively motile, plasma membrane integrity, incapacitation, intact acrosome and fertility of half blooded Boer buck liquid semen. Ten ejaculates were collected by artificial vagina every four days. The collected semen was divided into four aliquots in accordance to the treatments extender (0%, 4%, 5% and % CF3 in TEY based extender, v/v, respectively). The overall percentage of progressively motile sperm were significantly higher (P<0.05) in TEY + 6% CF3 as compared to control and 4% CF3 but was not significantly different as compared to 5% CF3. The overall percentages of sperm with intact plasma membrane was significantly higher (P<0.05) in TEY based extender + 6% CF3 as compared to the control, 4% and 5% CF3. The percentage difference between the incapacitated spermatozoa were significantly higher (P<0.05) for both in 5% and 6% CF3 since 0 to 96 hours stored at 5°C as compared to control and 4%. The difference between the overall mean percentages of spermatozoa with intact acrosome in TEY based extender incorporated with 5% and 6% CF3 were significantly higher (P<0.05) than control and 4% CF3. Pregnancy rates to AI using only preserved semen at 5°C for 96 hours, extended in TEY based extender + 6% CF3 was 44.68%. It is concluded that additional CF3 into TEY based extender increased preservation ability of extender in liquid form of buck semen up to 96 hours at 5°C in TEY + 6% CF3.

Effect of different diluents and storage time on field fertility of cooled ram semen after vaginal insemination

In a field experiment, 206 ewes were inseminated vaginally using liquid semen collected from 7 rams two times with 52h intervals between sessions. Ejaculates were pooled, split into three equal parts and diluted with a milk-based extender (M), a Tris–glucose (T) and a Tris–glucose containing 0.03125% caproic acid (TC). Ejaculates from the first collection were cooled up to 5°C over 2h, incubated for 52h at 5°C and inseminated (M52, T52 and TC52), but ejaculates from the second collection were diluted and inseminated immediately (M0, T0 and TC0). The inseminations of ewes were performed after synchronizing estrus. Only ewes in estrus were inseminated. The results of the pair-wise comparison of the lambing rates between the six groups showed significant higher results for TC52 (69.6%) compared with TC0 (46.15%) and also T52 (41.6%; P<0.05). Consequently, adding caproic acid to Tris–glucose may be useful for ram semen if semen is stored for 52h at 5°C.

PENGARUH PENAMBAHAN a-TOKOFEROL PADA MEDIA PENGENCER TRIS KUNING TELUR TERHADAP KUALITAS SEMEN CAIR DOMBA GARUT

The study was conducted to know the influence of a- tocoferrol additional into the egg yolk tris extender medium to the liquid semen quality of “Garut” lamb (Ovis aries). Parameter which had been evaluated i.e. percentages of progressive motile sperm, percentages of viable sperm and percentages of plasma membrane. On the 4 th day evaluation showed that the addition of a- tocoferrol with the dose 0,04 g/100 ml into the egg yolk tris extender produced the highest percentages ofprogressive motile sperm, percentages of viable sperm and percentages of plasma membrane ( 49,0 ± 6,5%; 68,6 ± 4,2% and 59,3 ± 6,5% ) but there were not significantly different from control ( 45,5 ± 5%; 66,0 ± 4,6% and 56,2 ± 5,7% )and the addition of a- tocoferrol with the dose 0,02 g/100 ml ( 49,0 ± 6,5%; 66,6 ± 4,0% and 59,3 ± 2,8% ) respectively. In conclusion, the addition of a-tocoferrol in the egg yolk tris extender medium no significant effect on the liquid semen quality.

Effect of oxytocin treatment on the reproductive performance of sows after artificial insemination with liquid semen

This study was designed to evaluate the effect of oxytocin treatment at the time of insemination on fertility and prolificacy of sows during the summer season (July and September). The study included 107 Polish Large White × Polish Landrace sows divided into 3 groups: C, control (without oxytocin); I, experimental (5 IU oxytocin administrated intramuscularly per sow, 2 min before insemination); and II, experimental (5 IU of oxytocin added into a 100-mL seminal dose, at the time of insemination). Semen was collected from 11 boars: 2 Polish Large White, 5 Polish Landrace, and 4 Duroc × Pietrain. The highest farrowing rate was obtained in group II at 94.4%. Compared with groups C at 63.9% and I at 71.43%, the demonstrated differences were statistically significant (P ≤ 0.01). The interaction between oxytocin and the breed of boar was also demonstrated. The highest significant differences were observed for Polish Landrace boars semen from group II compared with that of group C (P =

218 ANTIBIOTIC-RESISTANT MICROBIAL CONTAMINATION (ENTEROBACTER CLOACAE) DERIVED FROM EGG YOLK, FROZEN SEMEN EXTENDER, IN PORCINE IN VITRO-FERTILIZED EMBRYOS

The semen storage medium and the embryo culture environment are important for the further development of pre-implantation embryos, and these environments provide an ideal habitat for the propagation of a variety of microorganisms. Even though all precautions are taken to prevent contamination, this happens frequently during IVF and in vitro culture (IVC). Therefore, the aim of the present study was to investigate the source of contamination during semen processing for in vitro uses. In the present study, frozen semen was prepared from liquid semen (liquid semen was prepared according the method of (Weitze 1990 Reprod. Dom. Anim., 231–253 suppl. 1), and purchased from the Veterinary Service Laboratory, Department of Livestock Research (Yongin city, Gyeonggi-do, Korea) in our laboratory for IVF experiments because of a lack of fresh semen. Antibiotics were added to the frozen semen extender (kanamycin and gentamicin) and IVC medium (gentamicin) to further inhibit the growth of microorganisms. Nevertheless, microorganisms were observed to proliferate in the IVC drop when culturing IVF embryos using frozen semen. Three samples were randomly taken from the liquid semen, frozen semen, and egg yolk. Contaminated IVC medium, frozen–thawed semen, liquid semen, and egg yolk were cultured in de Man, Rogosa, and Sharpe agar medium. Identical whitish colonies were detected in the contaminated IVC drop, frozen–thawed semen samples, and egg yolk, but no colonies were formed in liquid semen samples. Identical gram-negative and rod-shaped bacteria were found in both the frozen–thawed semen sample and the contaminated IVC drop and egg yolk samples. Enterobacter cloacae were confirmed by API 20E kit according to the manufacturer’s instructions, with an identification value of 94.3% and a T index of 0.88. Antibiotic susceptibility tests were done according to CLSI (Wayne, PA) by using an ampicillin, amikacin, cephalothin, gentamicin, kanamycin, tetracycline, oxytetracycline, sulfamethoxazole, trimethoprim, norfloxacin, and ciprofloxacin test. Among them, E. cloacae were resistant to ampicillin, amikacin, cephalothin, gentamicin, and kanamycin but were susceptible to tetracycline, oxytetracycline, sulfamethoxazole and trimethoprim, norfloxacin, and ciprofloxacin. We suggest, based on these findings, that the sources of contamination might be from egg yolk. Therefore, synthetic semen extenders, which are free of egg yolk, might be considered during semen preparation. This work was supported by a grant (20070301034040) from the BioGreen 21 Program, Rural Development Administration, Republic of Korea.

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.