Influence of Equine Conformation on Rider Oscillation and Evaluation of Horses for Therapeutic Riding

To obtain basic knowledge about selecting horses for therapeutic riding, the influence of equine conformation on rider oscillation and relationships between these factors and the evaluation on horses as the therapeutic riding were studied. Thirty-five riding horses were used. Equine conformation was estimated by 24 indices. Rider oscillation was measured by an accelerometer fixed at the rider’s waist. The spatial position of the oscillation was estimated by a double integration of the acceleration. Horses were evaluated for therapeutic riding by a Riding for the Disabled Association instructor as a rider. Evaluations were on a scale of 1 to 5, with 5 being the highest score for 27 items. Horses were classified into 4 groups: the short and narrow (SN), short and wide (SW), tall and narrow (TN), and tall and wide (TW). The frequencies of rider oscillation both at walk and trot were higher (P<0.01), and the vertical (P<0.01) and longitudinal (P<0.05) amplitudes at trot were smaller, on short horses than on tall horses. The vertical amplitude at walk was smaller (P<0.05) and the lateral amplitude at trot was larger (P<0.01) on wide horses than on narrow horses. Short horses could be used for the rider who requires side walkers. Wide horses could be used for relieving muscular tension and for the rider who could not maintain good balance on the horse. Short and wide horses should be suitable for therapeutic riding.     

Effects of Bedding Material on the Lying Behavior in Stabled Horses

The objective of this study was to investigate the effect of straw, sawdust, coconut husk (husk), and coconut fiber (fiber) on the welfare of stable horses by observing their resting behavior. Twenty horses with ages ranging from 3 to 21 years were used at the Equine Research Institute of the Japan Racing Association, Utsunomiya, Japan. Five horses were allocated to each bedding condition. The behavior of each horse was recorded by video camera for 3 days and was continuously sampled from 17:00 to 05:00. The total duration, the number of bouts, and the mean and the maximum duration of bouts in standing rest, sternal lying, and lateral lying were calculated and analysed by the Kruskal-Wallis test and post hoc Steel-Dwass test. There was no difference in the standing rest and the sternal lying among beddings. Significant differences were observed in these values in the lateral lying among the different beddings (P<0.05). The values of the means of the total duration, the number of bouts, and the mean and the maximum duration of bout in the lateral lying were greater when husk was used as the bedding material than when sawdust were used (P<0.05). The results of the observations show that the new bedding materials would be as usable as straw. However, lateral lying was observed less frequently when sawdust were used as bedding; this indicates that use of sawdust as bedding material will decrease the welfare of stabled horses.     

Effect of a novel rice fermented extract on gastric ulcers in horses

Gastric ulcers cause appetite loss, poor body condition, and colic in horses. This study investigated the protective effect of a rice fermented extract on the gastric mucosa in 17 healthy Thoroughbreds. For one month, horses in the rice fermented extract (nine horses) and control (eight horses) groups were orally administered a rice fermented extract (100%; 0.2 ml/kg, SID) and tap water (0.2 ml/kg), respectively. Gastric endoscopic images were obtained before and one month after rice fermented extract administration. The gastric ulcer score was lower after administration (median, 1; maximum, 2; minimum, 1) than before administration (median, 4; maximum, 4; minimum, 3) in the rice fermented extract group (P<0.05). In conclusion, the administration of a rice fermented extract for one month improves gastric mucosal lesions in Thoroughbreds with gastric ulcers.     

Comparison of Ethylene Glycol and Propylene Glycol for the Vitrification of Immature Porcine Oocytes

Our aim was to optimize a cryoprotectant treatment for vitrification of immature porcine cumulus-oocyte complexes (COCs). Immature COCs were vitrified either in 35% ethylene glycol (EG), 35% propylene glycol (PG) or a combination of 17.5% EG and 17.5% PG. After warming, the COCs were in vitro matured (IVM), and surviving oocytes were in vitro fertilized (IVF) and cultured. The mean survival rate of vitrified oocytes in 35% PG (73.9%) was higher (P<0.05) than that in 35% EG (27.8%). Oocyte maturation rates did not differ among vitrified and non-vitrified control groups. Blastocyst formation in the vitrified EG group (10.8%) was higher (P<0.05) than that in the vitrified PG group (2.0%) but was lower than that in the control group (25.0%). Treatment of oocytes with 35% of each cryoprotectant without vitrification revealed a higher toxicity of PG on subsequent blastocyst development compared with EG. The combination of EG and PG resulted in 42.6% survival after vitrification. The maturation and fertilization rates of the surviving oocytes were similar in the vitrified, control and toxicity control (TC; treated with EG+PG combination without cooling) groups. Blastocyst development in the vitrified group was lower (P<0.05) than that in the control and TC groups, which in turn had similar development rates (10.7%, 18.1% and 23.3%, respectively). In conclusion, 35% PG enabled a higher oocyte survival rate after vitrification compared with 35% EG. However, PG was greatly toxic to oocytes. The combination of 17.5% EG and 17.5% PG yielded reasonable survival rates without toxic effects on embryo development.     

Change in Morphology of Spermatozoa from Dismount Semen during the Breeding Season in Thoroughbred Stallions in Japan

To clarify the physiological changes of sperm morphology in active Thoroughbred stallions during the breeding season, we examined the dismount semen collected from the penile urethra immediately after service. The spermatozoa were analyzed for relationships between the morphology and the stallion’s age or the number of services. Seasonal variation was apparent in the rate of the sperm tail abnormalities, spermatozoa with cytoplasmic droplets, appearance of medusa cells, and sperm head length. Area and width of the sperm head correlated negatively with age (P<0.05). The rate of appearance of medusa cells and the length of the sperm head were positively related to the number of services (P<0.05), and the aspect ratio was negatively related (P<0.01).     

Effects of ozonated autohemotherapy on the antioxidant capacity of Thoroughbred horses

The performance of horses undergoing regular intense exercise is adversely affected by oxidative stress. Thus, it is important to increase antioxidant production in horses in order to reduce oxidative stress. Ozonated autohemotherapy (OAHT) reportedly promotes antioxidant production. This study aimed to evaluate the effects of OAHT on antioxidant capacity. Ten Thoroughbred horses were used in this study. After the OAHT, we collected serum samples and measured biological antioxidant potential (BAP). We found that BAP began to increase after the OAHT and was significantly higher in the OAHT group than at 3 (P<0.01) and 7 days (P<0.05) after OAHT than in the control group at 3 and 7 days after starting collection of blood samples. Therefore, it was shown that OAHT improved the antioxidant capacity of the horses.     

Effect of 7,8-Dihydroxyflavone as an Antioxidant on In Vitro Maturation of Oocytes and Development of Parthenogenetic Embryos in Pigs

One of the factors that impairs in vitro produced porcine embryos is the oxidative stress that is mainly caused by the imbalance between reactive oxygen species (ROS) generation and antioxidants activity, especially that of glutathione (GSH). Here, we examined the effect of 7,8-dihydroxyflavone (7,8-DHF), a kind of flavonoid antioxidant, on porcine oocyte maturation and its developmental competence. Porcine oocytes were cultured in media supplemented with 0, 1, 5 and 10 μM 7,8-DHF during both in vitro maturation (IVM) and in vitro culture (IVC) after parthenogenetic activation. Maturation of oocytes was evaluated based on first polar body (PB) extrusion and intracellular GSH level, and developmental competence was assessed through observing cleavage and blastocyst formation. In each step, the levels of intracellular GSH and ROS were assessed by fluorescence intensity, and the apoptosis-related gene expression was examined using semiquantitative RT-PCR. The group treated with 1 μM 7,8-DHF during IVM and IVC showed increased cytoplasmic maturation and reached the blastocysts stage (36.1%) at a higher rate than the other groups (24.7, 16.0 and 10.3% for 0, 5 and 10 μM, P<0.05). In that group, the intracellular GSH level was significantly increased while ROS generation was significantly decreased after IVM and IVC (P<0.05). Moreover, it showed high expression of an anti-apoptotic gene (BCL2L1) and low expression of a pro-apoptotic gene (BAK1) (P<0.05). In conclusion, treatment with 1 μM 7,8-DHF during IVM and IVC showed an anti-apoptotic effect by increasing intracellular GSH synthesis and scavenging ROS and therefore improved the developmental competence of porcine embryos.     

Interrelationships Between Apoptosis and Fertility in Bull Sperm

Male fertility, the ability of sperm to fertilize and activate the egg and support early embryogenesis, is vital for mammalian reproduction. Despite producing adequate numbers of sperm with normal motility and morphology, some males suffer from low fertility whose molecular mechanisms are not known. The objective was to determine apoptosis in sperm from high and low fertility bulls and its relationship with male fertility. DNA damage, phosphatidylserine (PS) translocation, and expression of pro- and anti-apoptotic proteins (BAX and BCL-2) in the sperm were determined using TUNEL, Annexin V, and immunoblotting approaches, respectively. Amounts of apoptotic spermatozoa were 2.86 (± 1.31) and 3.00 (± 0.96) in high and low fertility bulls, respectively (P=0.548), and were not correlated with fertility. There was a negative correlation between early necrotic spermatozoa and viable spermatozoa (r = –0.99, P<0.0001). Fertility scores were correlated with live spermatozoa detected by eosin-nigrosin test and necrotic spermatozoa determined via flow cytometry (r = –0.49, P<0.006 and r = –0.266, P<0.0113, respectively). BAX level was higher in low fertile group than high fertile group; however, this difference was not statistically significant due to the variations of bull samples (Bull 1–3 vs. Bull 4–5) in low fertile group (P<0.283). BCL-2 was not detectable in any of the sperm samples. The results shed light onto molecular and cellular underpinnings of male fertility.     

Persistence of virus-neutralizing antibodies in horses inoculated with two doses of a live equine herpesvirus type 1 vaccine with different vaccination intervals

The antibody response in horses inoculated with 2 doses of a live equine herpesvirus type 1 vaccine with different vaccination intervals (1 to 3 months) was evaluated with regard to the persistence of virus-neutralizing (VN) antibodies. The durations for which the geometric mean VN titers were maintained significantly higher than those before the first vaccination (P<0.05) were up to 5 months in horses that received the vaccination with a 1-month interval (n=17) and 7 months for those that received it with a 2-month (n=17) or 3-month interval (n=14 or 17). The vaccination program with the 2-month interval was the most effective in maintaining VN antibodies for a long duration with the smallest gap of antibody decline between the first and second vaccinations.     

Good Thermally Conducting Material Supports Follicle Morphologies of Porcine Ovaries Cryopreserved with Ultrarapid Vitrification

Effects of supporting materials during vitrification procedure on the morphologies of preantral follicles of pig ovaries were assessed. Ovarian cortical sections of prepubertal pigs were randomly allocated to 5 groups. The sections were vitrified ultrarapidly with 5 different vitrification devices. The sections were put on 4 fine needles (Cryosupport), on a thin copper plate, or on a carbon graphite sheet or were sandwiched between copper plates or between carbon graphite sheets before cooling. The cooling and warming rates with the graphite sheets were significantly higher than those with the copper plates (P<0.05). A total of 3,064 follicles were analyzed following HE staining after vitrification with 5 different devices. The morphologies follicles vitrified on the Cryosupport or on the graphite sheet were well preserved compared with those vitrified on the copper plate or between copper plates (P<0.01). The morphologies of follicles vitrified between copper plates were mostly damaged (P<0.05). Taken together, good thermally conducting material supports follicle morphologies of ovaries cryopreserved with ultrarapid vitrification.     

Progesterone Profiles in the Caudal Vena Cava and Jugular Vein in Response to Pulsatile Luteinizing Hormone Stimulation Induced by GnRH Treatment During the Mid-luteal Phase in Lactating Dairy Cows

The aim of this study was to examine whether increased frequency of luteinizing hormone (LH) pulses influences luteal progesterone (P₄) secretion by measuring progesterone concentrations at the secreted (caudal vena cava) and circulating levels (jugular vein) in lactating dairy cows. Cows received six intravenous administrations of 2.5 μg of GnRH (gonadorelin acetate, n=4) or 2 ml saline (n=3) at 1-h intervals on 12.4 ± 0.4 (mean ± SE) days after ovulation. Blood samples were collected from the caudal vena cava and jugular vein every 12 min for 12 h (6 h before and after treatment). During the 6 h after treatment, frequency of LH pulses (5.3 ± 0.3 and 3.0 ± 0.0 pulses/6 h) and mean LH concentration (0.50 ± 0.06 and 0.38 ± 0.05 ng/ml) were greater (P<0.05) in GnRH-treated cows than in saline-treated cows. Mean P₄ concentration and amplitude of P₄ pulses in the caudal vena cava during the 6 h after treatment were greater (P<0.05) in GnRH-treated cows than in saline-treated cows, but the frequency of P₄ pulses was not different between the groups. Mean P₄ concentration in the jugular vein during the 6 h after treatment was also higher (P<0.05) in GnRH-treated cows than in saline-treated cows (7.0 ± 1.3 and 5.4 ± 0.9 ng/ml). These results indicate that the increased frequency of LH pulses stimulates progesterone secretion from the functional corpus luteum and brings about higher P₄ concentrations in the circulating blood in lactating dairy cows.     

Ascorbic Acid Improves the Developmental Competence of Porcine Oocytes After Parthenogenetic Activation and Somatic Cell Nuclear Transplantation

In this study, a dose-response assessment was performed to understand the relation between supplementation of media with L-ascorbic acid or vitamin C and porcine oocyte maturation and the in vitro development of parthenotes (PA) and handmade cloned (HMC) embryos. Various concentrations (0, 25, 50 and 100 µg/ml) of vitamin C supplemented in in vitro maturation (IVM) and culture (IVC) media were tested. None of these vitamin C additions affected nuclear maturation of oocytes, yet supplementation at 50 µg/ml led to significantly increased intracellular glutathione (GSH) levels and reduced reactive oxygen species (ROS). When cultured in IVM- and/or IVC-supplemented media, the group supplemented with 50 µg/ml of vitamin C showed improved cleavage rates, blastocyst rates and total cell numbers per blastocyst (P<0.05) compared with other groups (control, 25 µg/ml and 100 µg/ml). In contrast, supplementation with 50 µg/ml vitamin C decreased (P<0.05) the apoptosis index as compared with the groups supplemented with 100 µg/ml. In addition, even with a lower blastocyst rate to start with (37.6 vs. 50.3%, P<0.05), supplementation of HMC embryos with vitamin C ameliorated their blastocyst quality to the extent of PA embryos as indicated by their total cell numbers (61.2 vs. 59.1). Taken together, an optimized concentration of vitamin C supplementation in the medium not only improves blastocyst rates and total cell numbers but also reduces apoptotic indices, whereas overdosages compromise various aspects of the development of parthenotes and cloned porcine embryos.     

Treatment with a High Dose Combination of PMSG/hCG Preparation of Mares Clinically Diagnosed with Ovarian Quiescence during the Breeding Season (Investigation from 1975 to 2000)

A total of 88 thoroughbred mares were diagnosed with clinical ovarian quiescence and subjected to four treatment regimens. Using PMSG, hCG or combinations of both. A high dose combination of 5,000IU PMSG with 5,000IU hCG showed significantly higher rates of marked estrus and ovulation induction (P<0.01) as well as conception rates (P<0.05). In the present study, the administration of a high-dose combination of PMSG with hCG was shown to be an effective treatment of ovarian quiescence in light mares.     

Genetic variants of the unsaturated fatty acid receptor GPR120 relating to obesity in dogs

G protein-coupled receptor (GPR) 120 is an unsaturated fatty acid receptor, which is associated with various physiological functions. It is reported that the genetic variant of GPR120, p.Arg270His, is detected more in obese people, and this genetic variation functionally relates to obesity in humans. Obesity is a common nutritional disorder also in dogs, but the genetic factors have not ever been identified in dogs. In this study, we investigated the molecular structure of canine GPR120 and searched for candidate genetic variants which may relate to obesity in dogs. Canine GPR120 was highly homologous to those of other species, and seven transmembrane domains and two N-glycosylation sites were conserved. GPR120 mRNA was expressed in lung, jejunum, ileum, colon, hypothalamus, hippocampus, spinal cord, bone marrow, dermis and white adipose tissues in dogs, as those in mice and humans. Genetic variants of GPR120 were explored in client-owned 141 dogs, resulting in that 5 synonymous and 4 non-synonymous variants were found. The variant c.595C>A (p.Pro199Thr) was found in 40 dogs, and the gene frequency was significantly higher in dogs with higher body condition scores, i.e. 0.320 in BCS4–5 dogs, 0.175 in BCS3 dogs and 0.000 in BCS2 dogs. We conclude that c.595C>A (p.Pro199Thr) is a candidate variant relating to obesity, which may be helpful for nutritional management of dogs.     

Cardiac arrhythmias and electrolyte disturbances in colic horses

Background

Despite increased focus on cardiac arrhythmias in horses, the nature and prevalence is still poorly described. Case reports suggest that arrhythmias occurring secondary to systemic disease are seen more commonly in the clinic than arrhythmias caused by cardiac disease. The aim of this study was to investigate the prevalence of arrhythmias in colic horses referred for hospital treatment. Associations between electrolyte disturbances and arrhythmias were also investigated.The study population consisted of eight control horses and 22 referred colic horses. A Holter electrocardiography (ECG) was recorded during the first 24 hours of admission. The ECG’s were analysed by a software program followed by manual visual inspection. Arrhythmias registered included second degree atrioventricular (AV) blocks, supraventricular premature complexes (SVPCs), and ventricular premature complexes (VPCs). Blood was collected at admission and again between 12 and 24 hours after ECG was applied, and analysed for concentrations of potassium, sodium, ionised calcium, chloride, glucose, and L-lactate.

Results

Heart rate was 37.4 ± 3.7 bpm in the control group, and 51.6 ± 11.8 bpm, in the colic group, which was significantly different (P < 0.0001). AV blocks and SVPCs were found in both groups, however only colic horses showed VPCs. No significant difference between the two groups was found for AV blocks, SVPCs, and VPCs (P = 0.08 - 0.76). The mean levels of potassium, sodium, ionized calcium, and chloride were significantly lower in the colic group compared to the control group at admission. Mean levels of glucose and L-lactate were significantly elevated in the colic group (P < 0.05).

Conclusions

This study describes prevalence of cardiac arrhythmias and electrolytes concentrations in colic horses compared to healthy controls. Although we only observed VPCs in the colic horses, no significant differences between colic horses and controls were found. Despite the colic horses having electrolyte changes at admission no correlation was found between the electrolyte disturbances and cardiac arrhythmias. Although no clear conclusions can be drawn from the present study, the results indicate that relatively mild colic per se is not pro-arrhythmogenic, whereas severe colic probably are more likely to result in ventricular arrhythmia.     

Endoscopic findings of the stomach in pleasure horses in Poland

Background

This study was performed to determine the prevalence of ulcers in the gastric squamous and glandular mucosa in Polish pleasure horses.

Study design

Medical records from gastroscopic examinations of 108 pleasure horses of different breeds were reviewed. The study population consisted of two groups; group I (n = 48) with horses that expressed mild clinical signs of gastric ulcer syndrome (EGUS) including poor appetite, slight weight loss or poor body condition, and group II (n = 60) with horses that had no signs of gastrointestinal problems. The age range was 4–10 years, including 5 males, 34 castrated males (geldings) and 69 mares. The prevalence, distribution and severity of gastric ulcers were recorded. Lesions involving the squamous mucosa and the glandular mucosa of the antrum and pylorus were graded and compared between groups.

Results

Significant difference was found in the presence and severity of gastric ulcers between the two groups of horses. The overall prevalence of gastric ulcers in the first group of horses (n = 48) was 59% while in the group of clinically healthy horses (n = 60) the prevalence of gastric lesion was 40% (P = 0.004). Almost 19% of horses from group I had between 6–10 lesions (EGUS score III) and nearly 19% had either >10 localized lesions or very large diffuse lesions (EGUS number score IV). The number of ulcerations in affected horses were significantly lower in group II compared to group I (P = 0.016) as 10% of horses had 6–10 lesions (EGUS number score III) and nearly 14% had either >10 localized lesions or very large diffuse lesions (EGUS number score IV). Gastroscopy revealed that nearly 32% of horses from the second group had an ulceration EGUS score ≥ II.

Discussion and conclusions

This study confirms that gastric ulcerations can be prevalent in apparently clinically normal pleasure horses and a complete gastroscopic examination including the examination of the pylorus is advisable to evaluate this syndrome.     

Lipoprotein cholesterol and triglyceride concentrations associated with dog body condition score; effect of recommended fasting duration on sample concentrations in Japanese private clinics

The objectives of this study were to survey clinics’ guidance about recommended fasting duration (FD) prior to lipoprotein analysis, and to characterize lipoprotein cholesterol and triglyceride concentrations in obese and overweight dogs categorized on the basis of the 5-point body condition score (BCS) scale. A dataset was created from lipoprotein analysis medical records of 1,538 dogs from 75 breeds in 354 clinics from 2012 to 2013. A phone survey was conducted to obtain the clinics’ FD. Two-level linear mixed-effects models were applied to the data. Over 50% of the clinics said they recommended fasting for 12 hr or more. Dogs in clinics with FD 12 hr or more had lower chylomicron triglyceride concentrations than those in clinics with FD less than 8 hr (P=0.05). Mean (± SEM) BCS at sampling was 3.7 ± 0.02. Obese and overweight dogs had higher very low density lipoprotein (VLDL) and high density lipoprotein (HDL) cholesterol and triglyceride concentrations than ideal dogs (P<0.05), but no such difference was found for low density lipoprotein cholesterol and triglyceride concentrations (P≥0.07). Across all BCS, as dog age rose from 0 to 8 years old, HDL cholesterol concentrations decreased by 13.5 mg/dl, whereas VLDL triglyceride concentrations increased by 81.7 mg/dl (P<0.05). In conclusion, FD of 8 hr or less may affect lipoprotein lipid concentrations. Obese and overweight dogs were characterized as having high VLDL and HDL cholesterol and triglyceride concentrations.     

Production of Functional Gametes from Cryopreserved Primordial Germ Cells of the Japanese Quail

The Japanese quail (Coturnix japonica) is a valuable bird as both an experimental animal, for a wide range of scientific disciplines, and an agricultural animal, for the production of eggs and meat. Cryopreservation of PGCs would be a feasible strategy for the conservation of both male and female fertility cells in Japanese quail. However, the effects of freeze-thaw treatment on viability, migration ability and germline transmission ability of quail PGCs still remain unclear. In the present study, male and female PGCs were isolated from the blood of 2-day-old embryos, which were cooled by slow freezing and then cryopreserved at –196 C for 77–185 days, respectively. The average recovery rate of PGCs after freeze-thawing was 47.0%. The viability of PGCs in the frozen group was significantly lower than that of the control group (P<0.05) (85.5% vs. 95.1%). Both fresh and Frozen-thawed PGCs that were intravascularly transplanted into recipient embryos migrated toward and were incorporated into recipient gonads, although the number of PGCs settled in the gonads was 48.5% lower in the frozen group than in the unfrozen control group (P<0.05). Genetic cross analysis revealed that one female and two male recipients produced live progeny derived from the frozen-thawed PGCs. The frequency of donor-derived offspring was slightly lower than that of unfrozen controls, but the difference was not significant (4.0 vs. 14.0%). These results revealed that freeze-thaw treatment causes a decrease in viability, migration ability and germline transmission ability of PGCs in quail.     

Patient factors influencing the concentration of stromal vascular fraction (SVF) for adipose-derived stromal cell (ASC) therapy in dogs

The objective of this study was to determine whether patient factors influence the concentration of the stromal vascular fraction (SVF) in fat for adipose-derived stromal cell (ASC) therapy in dogs. A total of 1265 dogs underwent adipose collection surgeries by veterinarians for processing by the Vet-Stem laboratory and data on cell counts and patient factors were collected. Body condition score (BCS) and breed size did not significantly affect the viable cells per gram (VCPG) of adipose tissue that represents the viable SVF. Age significantly affected the VCPG, with dogs in age quartile 1 having a significantly higher VCPG than those in quartile 2 (P = 0.003) and quartile 4 (P = 0.002). Adipose tissue collected at the falciform location had significantly fewer VCPG than tissue collected at the thoracic wall and inguinal locations (P < 0.001). When the interaction of gender and location was evaluated, there were significantly fewer VCPG in tissue collected at the falciform location than at the thoracic wall and inguinal locations in female spayed dogs (P < 0.001) and male neutered dogs (P < 0.001), but not in female intact dogs (P = 0.743) or male intact dogs (P = 0.208). It was concluded that specific patient factors should be taken into consideration in order to obtain the maximal yield of VCPG from an adipose collection procedure.     

Sperm Pretreatment with Dithiothreitol Increases Male Pronucleus Formation Rates After Intracytoplasmic Sperm Injection (ICSI) in Swamp Buffalo Oocytes

Failure of male pronucleus formation has hampered the success of intracytoplasmic sperm injection (ICSI) in swamp buffalo. The aim of the present study was to improve male pronucleus formation by pretreating sperm with various chemicals before ICSI. In Experiments1 and 2, sperm were treated according to one of the following protocols: (1) 0.1% Triton-X 100 (TX) for 1 min, (2) 10 µM calcium ionophore (CaI) for 20 min, (3) freezing and thawing (FT) without any cryoprotectant, or (4) no treatment (control). These sperm treatment groups then either did or did not receive additional sperm treatment with 5 mM dithiothreitol (DTT) for 20 min. Acrosomal integrity (Experiment 1) and DNA fragmentation (Experiment 2) were evaluated in the sperm before ICSI. In Experiment 3, oocytes matured in vitro were subjected to ICSI using pretreated sperm as described above and then were cultured either with or without activation. The TX- and CaI-treated sperm caused an increase in the number of acrosome-loss sperm, whereas the FT treatment and control increased the proportion of acrosome-reacted sperm (P<0.05). The DNA fragmentation did not differ among treatments (P>0.05). At 18 h post-ICSI, pronucleus (PN) formation was found only in activated oocytes. The majority of the activated ICSI oocytes contained intact sperm heads. Normal fertilization was observed in the CaI and FT treatment groups and control group when sperm were treated with DTT before ICSI. In conclusion, DTT treatment of sperm with reacted acrosomes before ICSI together with activation of the ICSI oocytes is important for successful male pronucleus formation.