Effects of extenders and cryoprotectants on cryopreservation of Thai red junglefowl (Gallus gallus gallus) spermatozoa.

Semen cryopreservation is crucial maintain genetic diversity of avian species. Little is known about suitable extenders for post-thaw survival of spermatozoa from Thai red junglefowl (Gallus gallus gallus). Therefore, the present study aimed to compare the suitability of the modified Thai red junglefowl extender (TRJFE) for cryopreservation of Thai red junglefowl spermatozoa with other extenders, including the Schramm extender, the red junglefowl extender (RFE), and the HS1 extender, in terms of sperm viability, motility, and fertility. First, the effects of adding 6% and 9% (v/v) N,N-dimethylacetamide, N,N-dimethylformamide (DMF), or N-methyl acetamide to these extenders on the post-thaw sperm quality of pooled ejaculates from 25 male fowls. The viability of thawed sperm was assessed by using nigrosin-eosin staining and sperm motility was determined by using computer-assisted sperm analysis. Fertility was assessed by inseminating 144 laying hens. The TRJFE +6% DMF combination significantly improved post-thaw viability (64.88 ± 0.51%) and motility (68.58 ± 1.13%) of Thai red junglefowl sperm, and the semen had the highest fertility (60.97 ± 0.72%). The findings suggest that TRJFE +6% DMF is a suitable freezing medium to conserve Thai red jungle fowl genetic resources.

Effect of organic selenium dietary supplementation on quality and fertility of cryopreserved chicken sperm.

Oxidative stress due to cryopreservation has been considered as a major factor in sperm damage. Supplementation of the diet with different concentrations of organic selenium has been proposed to improve the quality of fresh and frozen-thawed semen in different breeds of roosters. Sixteen Pradu Hang Dum (Thai native) and 16 Rhode Island Red roosters were used in this study. Four levels of selenium supplementation between 0 and 0.9 ppm were examined. After 14 days of feeding, semen samples were collected twice a week and the fresh semen was evaluated. Then semen from each group was pooled and cryopreserved. The fertility of frozen-thawed semen was determined by inseminating 48 layer hens. Supplementation of diets with 0.3, 0.6 and 0.9 ppm selenium improved the fresh semen in terms of sperm viability and normal morphology (P < 0.01). Sperm concentration increased (quadratically, P < 0.001) with increasing dietary selenium levels. Meanwhile, post-thawed semen quality in terms of sperm motility, viability, live with intact acrosome and functioning mitochondria improved significantly with selenium treatments of 0.6 and 0.9 ppm, and lipid peroxidation was decreased (P < 0.001) and fertility improved (P < 0.05) with those levels of selenium treatment. In addition, there were differences between breeds with respect to some fresh or frozen semen quality parameters (P < 0.05). In conclusion, the breed affected both fresh and frozen semen. Even there were no statistically significant differences in the parameters from groups 0.6 and 0.9 ppm on frozen-thawed semen quality, but the highest sperm concentration was found in 0.6 ppm. Therefore selenium supplementation of diets at 0.6 ppm was recommended to improve the quantity and quality of fresh and frozen semen.

Lipid profile of sperm cells in Thai native and commercial roosters and its impact on cryopreserved semen quality.

Plasma membrane lipids are the key factor in the ability of chicken sperm to be frozen. They ensure fluidity and flexibility of the sperm membrane for effective viability and motility during in vitro storage. The objective of this study was to compare the lipid profiles of different native and commercial chicken breeds: native Thai (Pradu Hang Dam) roosters and commercial (Rhode Island Red) roosters, with respect to their frozen sperm quality. In addition, lipid peroxidation and antioxidant enzymes superoxidase dismutase (SOD) and catalase (CAT) were also examined. Semen was collected from 12 roosters of each breed. For fresh semen, parameters assessed include semen volume, pH, sperm concentration, sperm motility, and viability, while for frozen semen, the parameters assessed were sperm motility and viability. Moreover, other parameters assessed included malondialdehyde (MDA) concentration, activities of SOD and CAT, and fatty acid profile. We found that sperm viability and motility of frozen semen were higher in the commercial breed than in the native breed (P < 0.05). The commercial chicken breed had higher MDA concentration than the native breed (P < 0.05), but antioxidant enzymes remained unchanged in both. Levels of arachidonic acid (AA; C20:4n-6) and docosahexaenoic acid (DHA; C22:6n-3) were significantly higher (P < 0.05) in the commercial than in the native breed; however, n-6 to n-3 ratios were not different. In conclusion, our study found that lipid profiles have an influence on frozen sperm viability and motility between the breeds. Polyunsaturated fatty acids, particularly AA and DHA, are beneficial to sperm quality.

Comparison of TNC and standard extender on post-thaw quality and in vivo fertility of Thai native chicken sperm.

Semen extender has a vital role in preservation of sperm cells properties in terms of sperm viability, motility, acrosome integrity, and mitochondrial membrane potential. The objective of the present study was to evaluate a new extender, known as Thai native chicken (TNC) extender compared to BHSV-based and modified Sasaki extenders for freezing chicken semen. Semen from Thai native roosters was collected, pooled, and randomly divided into three groups. Semen was frozen with a simple freezing method using nitrogen vapor and dimethylformamide. In the first experiment, post-thaw motion parameters, viability, acrosome integrity, mitochondrial function, and lipid peroxidation levels were analyzed using computer-assisted sperm analysis, propidium iodide, fluorescein isothiocyanate-conjugate peanut agglutinin, JC-1, and the thiobarbituric acid reaction. Results showed that the type of extender had no effect on the percentage of total motile and curvilinear velocity. The percentage of progressive motile, straight-line velocity, and average path velocity of post-thawed semen were significantly lower in TNC compared to the modified Sasaki extender. However, the percentages of post-thawed acrosome integrity and active mitochondria were significantly higher in TNC extender (P < 0.05). For the second experiment, semen was thawed by using each of extenders thereafter, was inseminated to 48-layer breeder hens to determine the fertility rate. Among the three extenders used, the highest fertility rate was found in TNC extender. In conclusion, TNC extender can be recommended as an appropriate and useful cryopreservation media for native chicken semen since it maintains the quality of rooster semen and fertility after freezing and thawing process.

Microtubule stabilisers docetaxel and paclitaxel reduce spindle damage and maintain the developmental competence of in vitro-mature bovine oocytes during vitrification.

This study compared the efficacy of docetaxel (DT) and paclitaxel (PT) in reducing spindle damage during vitrification and maintaining the developmental competence of in vitro-matured (IVM) bovine oocytes after vitrification and warming. Pretreatment of IVM oocytes with 0.05µM DT for 30min before vitrification resulted in significantly higher (P<0.05) rates of oocyte survival and cleavage after IVF, as well as subsequent blastocyst rates on Days 7-9 and hatching on Days 8-9, compared with oocytes pretreated with 1.0µM PT before vitrification or those vitrified without pretreatment. When nuclear status and spindle morphology of vitrified oocytes were assess after warming by immunostaining, DT pretreatment before vitrification resulted in a significantly higher (P<0.05) percentage of oocytes at the MII stage with a normal, intact spindle compared with PT pretreatment or no pretreatment, but the percentage of MII oocytes was still significantly lower (P<0.05) than in the control group. Pretreatment of IVM bovine oocytes with 0.05µM DT or 1.0µM PT for 30min before vitrification reduces spindle damage to the same extent, without side effects on fertilisation and development. Pretreatment with 0.05µM DT improved the developmental competence of vitrified-warmed oocytes to a greater degree than 1.0µM PT pretreatment.

Supplementing rooster sperm with Cholesterol-Loaded-Cyclodextrin improves fertility after cryopreservation.

Little is known about the effects of Cholesterol-Loaded Cyclodextrin (CLC) on post-thaw semen quality in chicken. The aim of the present study is to investigate the efficacy of CLC levels (0, 1, 2 and 3 mg/mL Schramm diluent) on post-thawed semen quality and fertility in two breeds of chicken Pradu Hang Dum (native chicken) and Rhode Island Red. Semen samples of each breed were pooled, divided into 4 aliquots and diluted with Schramm diluents, cooled to 5 °C when DMF was added (6% of final volume). Semen straws were subjected to cryopreservation using the liquid nitrogen vapor method. Post-thawed sperm motility, viability, acrosome integrity, mitochondrial function, and the Malondialdehyde (MDA) level were determined. The fertility of frozen semen was tested by inseminating laying hens. Post-thaw motility between Pradu Hang Dum and Rhode Island Red was no different; but Rhode Island Red had a higher semen viability and live cell intact acrosomes than Pradu Hang Dum (P < 0.05). The percentage of high functioning mitochondria in the Pradu Hang Dum was higher than the Rhode Island Red. CLC at 2 and 3 mg/mL supplementation was associated with improved viability of frozen semen; that is, acrosome integrity and mitochondrial function (P < 0.01), albeit having no effect on MDA levels. The sperm with 1 mg/mL CLC yielded a significantly better fertility (P < 0.01). CLC (1 mg/mL) improved the quality of frozen rooster semen. There was no interaction among breeds and CLC on post-thaw semen quality and fertility.

Comparison of three superovulation protocols with or without GnRH treatment at the time of artificial insemination on ovarian response and embryo quality in Thai native heifers.

To optimize the superovulation protocol in Thai native cattle, the present research was designed to (1) compare three different protocols designed to induce superstimulation and (2) study the effect of gonadotropin-releasing hormone (GnRH) administration at insemination time (to induce ovulation) on ovarian follicular activities in terms of the number of large follicles, corpora lutea (CLs) and unovulated follicles, and the number and quality of ova/embryos recovered in Thai native heifers. Initially, the estrous cycles of animals (n = 36) at unknown stages were synchronized by two prostaglandin F2α (PGF2α) injections at an interval of 12 days. Follicular development of heifers was randomly superstimulated with one of three different treatment protocols: treatment A-a total of 100 mg of pituitary-derived FSH (pFSH; Folltropin®-V) administered in eight decreasing doses; treatment B-a single dose of 100 mg pFSH dissolved in 30% (w/v) polyvinylpyrrolidone; or treatment C-ablation of all follicles ≥5 mm with a single dose of pFSH. All heifers received PGF2α 48 h after the initiation of FSH treatment to induce luteolysis from the previous cycle, and they were twice inseminated at 12 and 24 h after the onset of estrus. Heifers in each treatment were assigned to be injected or not with GnRH at the time of first insemination with frozen/thawed semen to induce ovulation. About 7 days after artificial insemination (AI), ova/embryos were collected and classified. The numbers of large follicles at the onset of estrus were not statistically significantly different; meanwhile, the maximum diameters of follicles at the time of first insemination in treatment C were smaller compared with the other treatment groups (p < 0.001). The administration of GnRH at the first insemination time resulted in a greater number of CLs and fewer unovulated follicles at the time of ova/embryo collection (p = 0.001), which subsequently resulted in a higher number of total ova/embryos recovered (p = 0.030). Among heifers treated with different superstimulation protocols, the ablation of all follicles ≥5 mm in diameter before superstimulation (treatment C) resulted in significantly higher quality of fertilized ova and transferable embryos (p = 0.001). In summary, it could be inferred that GnRH treatment improved ovarian function rather than embryo quality. Dominant follicle ablation prior to superstimulation is preferable for collecting a greater number of transferable embryos.

Pretreatment of in vitro matured bovine oocytes with docetaxel before vitrification: Effects on cytoskeleton integrity and developmental ability after warming.

The stabilization of spindle fibersis important for successful vitrification of bovine oocytes because microtubules and other cytoskeleton fibers (CSF) can be damaged during vitrification, resulting in failure of fertilization after thawing. Docetaxel, a stabilizing agent, could potentially reduce CSF damage of bovine oocytes induced during vitrification. However, there have been no reports on the effects of docetaxel on their vitrification. Experiment 1 was conducted to investigate the effects of various doses of docetaxel (0.0, 0.05, 0.5, 5.0 and 50 µM) in preincubation medium of in vitro matured (IVM) bovine oocytes on their developmental ability after in vitro fertilization (IVF). The results show that 0.05 µM docetaxel had no adverse effect on embryo development, while docetaxel at a concentration of ⩾0.5 µM inhibited development. Experiments 2 and 3 were conducted to investigate the effects of preincubation of IVM bovine oocytes with 0.05 µM docetaxel for 30 min prior to vitrification-warming on CSF integrity (Experiment 2), and on oocyte survival and viability after IVF (Experiment 3). When preincubated with 0.05 µM docetaxel for 30 min before vitrification, post-thawed oocytes had less CSF damage and higher survival rates compared with those untreated with docetaxel before vitrification. Surviving oocytes also had higher rates of cleavage and development to the blastocyst stage after IVF. In conclusion, preincubation of IVM bovine oocytes with 0.05 µM docetaxel for 30 min before vitrification was effective at preventing CSF damage during vitrification, and improving oocyte viability after warming and subsequent cleavage and blastocyst formation after IVF.

VITRIFICATION OF THAI NATIVE CATTLE OOCYTES: EFFECTS OF ETHYLENE GLYCOL CONCENTRATIONS AND EXPOSURE TIME, LINOLEIC ACID ALBUMIN AND CHOLESTEROL-LOADED METHYL-B-CYCLODEXTRIN.

The present study aimed to improve the oocyte vitrification procedure for preservation of Thai native cattle genetic resources. In Experiment I, oocytes were exposed to various doses (2%, 4% and 6%) of ethylene glycol (EG) in vitrification solution I (VS-I) for different equilibration times (10 or 20 min) before being exposed to VS-II and then subjected to vitrification. Experiment II was divided into two parts: (a) oocytes were matured in medium supplemented with linoleic acid albumin (LAA) (1% or 2%) and then vitrified; (b) matured oocytes were preincubated with cholesterol-loaded methyl-ß-cyclodextrin (CLC) (1% or 2%) and then vitrified. Equilibration of oocytes by exposure to 6% EG in VS-I for 10 min (Experiment I), and in vitro maturation of immature oocytes in medium supplementation with 2% LAA (Experiment II) were the most effective methods; vitrified/thawed oocytes showed higher rates of survival and subsequent embryonic development compared with the other experimental groups.

Cryopreservation of Sambar deer semen in Thailand.

Little is known of the different freezing and thawing techniques for post-thaw survival of spermatozoa in Sambar deer. So, this study determined the effect of seminal plasma, egg yolk and glycerol extenders and their concentrations, plus cooling, freezing, and thawing protocols on the post-thaw quality of their semen. Semen samples were collected by electro-ejaculation from four Thai Sambar deer stags (Cervus unicolor equinus). As evaluated by post-thaw progressive motility and acrosome integrity removal of seminal plasma was beneficial; Tris-egg yolk was the most efficient extender; a 20% egg yolk concentration was better than the 0%, 10%, or 30%; and a 3% glycerol concentration was better than 5%, 7%, or 9%. Using the optimum dilution techniques, semen was loaded in 0.5 ml plastic straws. Cooling times from ambient temperature to 5°C in 3 hr resulted in higher post-thaw progressive motility and acrosome integrity than 1, 2, or 4 hr. Suspending the straws 4 cm above the surface for 15 min before plunging into liquid nitrogen was better than suspending at 2 or 6 cm. For thawing frozen semen, an intermediate thawing (50°C, 8 sec) protocol was more effective than the slower (37°C, 10 sec) or faster (70°C, 5 sec) thawing rates. Timed insemination following estrus synchronization of 10 hinds resulted in six confirmed pregnancies at 60 days. Five hinds delivered live fawns. This study provides an effective approach for semen cryopreservation and artificial insemination (AI), which should be valuable to scientists for genetics and reproductive management of Sambar deer in developing countries.

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

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

Fertility and Insemination Characteristics of Sperm Storage Tubules in Old Thai-Native Hens.

We aimed to evaluate the effects of sperm concentration (150-250 × 106 spz/dose) and insemination frequency (once, twice, and thrice weekly) on fertility and sperm storage tubule (SST) characteristics. The SSTs were classified into five categories: namely, SSTs having an unscorable (SST1), empty (SST2), low (SST3), medium (SST4), and high (SST5) sperm count after insemination. The results showed that only insemination frequency affected the fertility rate (p < 0.05). The highest fertility was found in the thrice-weekly insemination group; however, this rate was not significantly different from that for the twice-weekly insemination group, except on day 7, while the once-weekly insemination group showed the lowest fertility rate (p < 0.05) from day four onward. On day 1, the SST characteristics showed no differences among the various insemination frequencies. On day 4, the SST2 and SST3 categories increased in the once-weekly insemination group (p < 0.05), while the SST4 and SST5 categories decreased compared to the twice- and thrice-weekly insemination groups (p < 0.05). On day 7, only the thrice-weekly insemination group maintained a level of SST5 category tubules like that measured on day 1 (p > 0.05). In summary, the insemination dose of 150 × 106 sperm was enough for fertilization, and thrice-weekly insemination was the appropriate frequency in old Thai native hens for maintaining a high sperm density in the SSTs throughout the week.

Superstimulation of follicular growth in Thai native heifers by a single administration of follicle stimulating hormone dissolved in polyvinylpyrrolidone.

This study was undertaken to determine whether a single i.m. injection of FSH dissolved in 10 ml of 30% (wt/vol) polyvinylpyrrolidone (PVP; MW=40,000) to form FSHp would induce follicular growth in Thai native heifers and to determine its optimal dose. In Group 1, heifers (n=4) were given multiple i.m. injections of FSHp every 12 h for 3 days at decreasing doses, for a total of 100 mg (control). In Groups 2, 3 and 4, heifers (n=4 in each group) were given single i.m. injections of FSHp at 50, 100 and 150 mg. All heifers received a single injection of 750 µg PGF2α 48 h after the initiation of exogenous FSH treatment. Ovaries of treated heifers were examined by transrectal ultrasonography every day until they showed estrus. Group 3 showed significantly higher numbers of ovulation follicles, significantly higher growth rates of follicles per day and significantly larger diameters of follicles and corpora lutea than groups 1 and 2 but not Group 4 (P<0.05). Group 4 showed significantly higher numbers of large follicles (≥5 mm in diameter), unovulated follicles and ovulations, a significantly higher growth rate of follicles per day, and significantly larger diameters of follicles and corpora lutea (P<0.05) than those of the other groups. This indicates a state of overstimulation of ovaries in this group. Besides, the plasma levels of FSH in Group 4 were significantly higher (P<0.05) than in the other group and were maintained in the range of 2.2-0.7 ng/ml over a period of 6 to 66 h after the FSHp injection. Meanwhile, the plasma levels of P4 and E2 did not differ in any of the groups in the period of 0 to 96 h during the superstimulation program. In conclusion, it was demonstrated that a single i.m. injection of 100 mg FSHp was the most effective dose for superstimulation of follicular growth in Thai native heifers under the experimental conditions in this study.

Solid storage supplemented with serine of rooster semen enhances higher sperm quality and fertility potential during storage at 5°C for up to 120 h.

Information on prolonging the storage duration of cold semen with acceptable fertility in roosters is limited. This study aimed to determine the efficiency of solid storage with the addition of various concentrations of serine to the Thai native rooster (Pradu Hang Dum) semen extender on semen quality and fertility potential during storage at 5°C for up to 120 h. Pooled semen was diluted with a base extender and a gelatin extender containing 0, 2, 4, and 6 mM serine, then stored at 5°C for 120 h. In Experiment 1, the semen quality and malondialdehyde (MDA) concentrations were assessed at 0, 24, 72, and 120 h after storage. In Experiment 2, fertility potential in terms of fertility and hatchability rates was determined using the most effective solid-storage semen from Experiment 1. Sperm quality decreased with increasing storage time (P < 0.05). The lowest semen quality was observed in the control group since T24 of storage compared with the other groups (P < 0.05). Progressive motility, viability, and mitochondrial function were higher (P < 0.05) in the extender supplemented with gelatin and serine groups than those in the gelatin alone group at T72 and T120. In the extender supplemented with gelatin and serine groups, the highest semen quality was observed in the gelatin with 4 mM serine groups. The differences among extenders supplemented with serine were insignificant (P > 0.05), and the lowest MDA was observed in the gelatin with 4 mM serine groups. The fertility and hatchability rates in gelatin with 4 mM serine at T24 were comparable to those in fresh semen (83.87 and 86.12% vs. 86.66 and 88.3%; P > 0.05). Those of T72 were significantly better than those of the control at the same hour of storage (64.08 and 71.61% vs. 52.38 and 64.48%), while those of T120 were not different among groups. In summary, a semen extender as a solid medium supplemented with 4 mM serine successfully preserved the rooster semen for a long duration up to 72 h of storage time.

Characterization of Oviduct Lining, with Emphasis on the Sperm Storage Tubule Region (Uterovaginal Junction), Correlated with Fertility in Mature and Old Thai Native Hens.

The effect of age on fertility was investigated in Thai native chickens. The objective of this study was to determine the effects of age (mature and old) on the morphological characteristics of the reproductive organs and the histological characteristics of the uterovaginal junction (UVJ) tissues, resident sperm in the UVJ, and fertility duration in Thai native hens. We found no differences in the morphological characteristics of the reproductive organs, except for the number of follicles and the sizes of the fifth large yellow follicle in mature hens, which were greater than those in old hens (p < 0.05). The diameter of the sperm storage tubules (SSTs) epithelium was larger in old hens than in mature hens (p < 0.05), whereas the epithelium height was lower in old hens (p < 0.05). The number of sperm in the SSTs was greater in mature hens compared with old hens (p < 0.05). Mature hens showed a higher fertility rate than old hens. Our results suggest that, in old hens, the function of the SSTs is impaired, and sperm cannot be retained. Such a deterioration of the SSTs may be one of the factors involved in the decline in fertility.

A novel route of follicle-stimulating hormone administration with a split-single ischiorectal fossa in Thai-Holstein crossbred superovulation programs under heat stress conditions.

The objectives of this study were to compare the efficiency of a split single injection of follicle-stimulating hormone (FSH) given by either intramuscular (split-single IM) or ischiorectal fossa (split-single IRF) injection to the traditional treatment and to determine the concentrations of FSH. The temperature and humidity index (THI) values were interpreted together with the ovarian responses and embryo characteristics. The ovarian responses in the split-single IRF group were similar to those of the control group (p > .05) but higher compared with the split-single IM group (p < .05). Higher peak levels of plasma FSH in the split-single IRF group did not differ compared with the control group (p > .05) but were lower in split-single IM administration (p < .05). The results showed a significant decrease in the numbers of large follicles and corpora lutea (CLs) in the moderate THI compared with low and high THI (p < .05). The high THI affected ovulation rate as well as the numbers of transferable embryos and degenerated embryos (p < .05). In conclusion, the split-single IRF administration had a comparable superovulatory response to the traditional twice-daily protocol. Moreover, the ovulation rate, ovarian follicle responses, and embryo quality were affected by heat stress.

Ovarian responses and FSH profiles at superovulation with a single epidural administration of gonadotropin in the Thai-Holstein crossbreed.

The conventional method of ovarian superstimulation requires multiple injections of gonadotropins which is time-consuming and may be stressful for the cows. This study was designed to determine whether a single epidural injection of FSH (EI group) would induce the superovulatory response in the Thai-Holstein crossbreed and evaluate FSH plasma hormone concentrations. Eight cows (replication = 3; n=24) were assigned to one of 2 treatments in switch back design. Control group (n=12): cows were received 400 mg FSH twice daily by intramuscularly for 4 days (80, 80, 60, 60, 40, 40, 20 and 20 mg), EI group (n=12): cows were received 400 mg FSH by single epidural injection. Data were collected in term of ovarian follicle responses, superovulatory responses, ova/embryo collection. FSH concentrations were examined using ELISA. The total follicular responses during oestrus were not different between treatments; however, the large follicles were less frequent (P < 0.01) while the medium follicle sizes were higher (P < 0.05) in the EI group. The plasma concentration of FSH in EI was dramatically increased within 2 hours before decreasing sharply thereafter (P < 0.01) and did not remain above baseline after 10 hours of administration. The embryo quality was better in the control than the EI groups (P < 0.05). Interestingly, the number of ovulation cysts in the EI group was 50%. The ovarian responses and embryo quality in the cows with cysts were worse compared with the non-cyst groups (P < 0.05). In conclusion, alternative protocols decreased the superovulatory response and increased poor embryo quality in Thai-Holstein crossbred. Also, the incidence of ovarian follicular cysts is higher in the EI group.

Different concentrations of cysteamine, ergothioneine, and serine modulate quality and fertilizing ability of cryopreserved chicken sperm.

The aim of this study was to evaluate the effects of freezing diluents supplemented in three potential amines/amino acids, namely, antioxidant cysteamine (2-aminoethanethiol [AET]), ergothioneine (ERG), and serine (SER), in optimization of chicken sperm cryopreservation. The semen of 36 Pradu Hang Dum males, selected based on their motility vigor score, was frozen by a simple freezing method using nitrogen vapors and dimethylformamide (DMF). In a first experiment, a wide range of AET, ERG, and SER doses were tested. Semen quality was evaluated after incubation at 5°C or after cryopreservation in straws in the Blumberger Hahnen Sperma Verdünner (BHSV) diluent + DMF (6% v/v) with or without AET, ERG, or SER. The best targeted doses of AET, ERG, or SER were then selected for experiment 2 that was focused on cryopreserved semen. Frozen-thawed sperm quality was evaluated by different in vitro tests and by evaluation of fertility. Objective motility parameters were evaluated by computer-assisted sperm analysis. Membrane integrity, acrosome integrity, and mitochondria function were evaluated using appropriate dyes and flow cytometry. Lipid peroxide production was assessed by the thiobarbituric acid test (malondialdehyde production). Fertility obtained with frozen-thawed semen supplemented or not in AET, ERG, or SER was evaluated after artificial insemination of laying hens. ERG and AET decreased sperm lipid peroxidation and decreased fertility, even at low doses. The presence of 4 mmol of SER significantly decreased lipid peroxidation, increased the frozen-thawed sperm quality, and increased fertility after sperm cryopreservation (90% vs. control 84%, P < 0.05). In a third experiment, the use of 1 mmol of sucrose (the best result of our previous study) added to 4 mmol of SER-supplemented extender was tested. This addition allowed to the highest levels of fertility (93%). In conclusion, the addition of 4 mmol of SER in semen cryopreservation diluents decreases peroxidation and improves the efficiency of the process.

Evaluation of cryopreserved boar semen after supplementation sericin form silkworm (Bombyx mori) in semen extender.

Boar cryopreserved semen is scarcely used for artificial insemination due to its quality which is largely reduced by membrane lipid peroxidation. This present study was designed to improve the post-thawed boar semen quality by determining the optimal level of sericin supplementation (antioxidants) in semen extender. Five levels of sericin supplementation between 0% and 1% (w/v) were examined. Semen was frozen by the liquid nitrogen vapor method, thawed slowly at 5°C for 5 min, and used for the evaluation of sperm quality. The results indicated 0.5%-1% sericin supplementation was more effective on maintenance of sperm viability, acrosome integrity, and mitochondrial functions during freezing-thawing. Moreover, 0.75% sericin supplementation was most protective toward total sperm motility and sperm progressive motility. Additionally, 0.25%-0.75% sericin supplementation significantly suppressed increases in the index of lipid peroxidation. In conclusion, 0.75% sericin is recommended as an alternative component of the freezing extender to improve cryopreserved boar semen. However, further research using AI will be necessary to demonstrate that this indication can be applied to the production of offspring in the farms.

Sucrose increases the quality and fertilizing ability of cryopreserved chicken sperms in contrast to raffinose.

Chicken semen conservation is an important tool for programs of genetic diversity management and of endangered breeds' conservation. However, the method still needs to be improved in order to be applied in a wide variety of environments and breeds. Our objective was to compare the effects of 2 external cryoprotectants saccharides (sucrose and raffinose) on the sperm freezability of a Thai local breed, Pradu Hang Dum, in which semen was frozen with a simple freezing method using nitrogen vapors and dimethyl formamide (DMF). Thirty-six males were selected on their motility vigor score for the experiments. In a first experiment, a large range of sucrose and raffinose doses were tested. Semen quality was evaluated after incubation at 5°C or after cryopreservation in straws in the saline Blumberger Hahnen Sperma Verdünner diluent + DMF (6% v/v) with or without sucrose/raffinose. The best targeted doses of sucrose and raffinose were then kept for experiment 2 that was focused on cryopreserved semen. In this experiment, semen quality was measured on frozen-thawed sperm: different objective motility data evaluated by computer-assisted sperm analysis (CASA), membrane integrity, acrosome integrity, mitochondria function evaluated using flow cytometry, lipid peroxide production assessed by the thiobarbituric acid test. Fertility obtained with frozen-thawed semen supplemented or not with sucrose or raffinose was also evaluated after artificial insemination of laying hens. The presence of sucrose at the osmotically inactive dose 1 mmol significantly increased the vigor motility, membrane integrity, acrosome integrity, and mitochondrial functions of frozen-thawed sperm (P < 0.05), and showed the highest levels of fertility after sperm cryopreservation (91% vs. control 86%, P < 0.001). Raffinose showed negative effects on in vitro semen quality from 1 to 100 mmol. Fertility was also negatively (P < 0.001) affected by raffinose (fertility rate 66 to 70%). We thus showed in the present study the high success of a simple chicken sperm cryopreservation method with an external cryoprotectant easily available and cheap, the sucrose, used at an osmotically inactive low concentration.