Luiz Ernandes Kozicki

Comparison between deep intracornual artificial insemination (dIAI) and conventional artificial insemination (AI) using low concentration of spermatozoa in beef cattle

This study aimed to compare the pregnancy rate using the conventional artificial insemination (AI) or deep intracornual artificial insemination (DIAI), with low number of spermatozoa (4.0 million sperm) in 270 Nelore cows. The animals were divided in two groups (G: G1 (135 cows) conventional AI was performed (=semen deposition in the uterine body) and in G2 (135 cows) to DIAI, in ipsilateral horn where the dominant follicle in the ovary had previously been detected, by ultrasound examinations. For both the methods, a single artificial insemination was carried out after visual estrus observation, checked three times a day (morning, afternoon and evening). The pregnancy diagnosis after 45 days was conducted by ultrasound. Results showed a better pregnancy rate in the DIAI group (67.4% - p<0.01), when compared to conventional AI (48.8%) with low spermatozoa concentration.

Viability of bull spermatozoa collected from the epididymis stored at 18-20°C

The aim of this study was to evaluate the viability of bull spermatozoa collected from the cauda epididymis stored at 18-20°C, which were compared with semen collected by electro-ejaculation method and preserved at 5°C. Ten pairs of testes from Tabapua bulls were removed by orchiectomy and stored for 6 (G6), 12 (G12), 18 (G18), 24 (G24) and 30 (G30) h at room temperature (18-20°C). Seven days before orchiectomy, semen was collected by electro-ejaculation method. The sperm parameters evaluated were: sperm motility, vigor, concentration, morphology and acrosome defects. Sperm motility declined (p<0.05) when spermatozoa were stored for 30 h in the epididymis. The spermatozoa from the epididymis showed lower sperm motility than that of spermatozoa collected via electro-ejaculation. There was a little expressive decrease in sperm vigor and increased in morphological defects with storage time, but the acrosome integrity was not affected. Cold storage (5°C) maintained sperm viable for 15 to 40.8 h. Thus, it was possible to recover viable sperm with 41.25% of motility from the cauda epididymis stored at room temperature of 18-20°C for 30 h. There were differences between the ejaculated and epididymal sperm for the bulls and the conservation at 5°C allowed short-term preservation of the gametes.

The effects of intramuscular or intravenous injections of gonadotropin releasing hormone at fixed-time artificial insemination on pregnancy rates of Bos indicus beef cows

The effect of an intramuscular versus intravenous administration of gonadotropin releasing hormone (GnRH) at fixed-time AI (FTAI) on the pregnancy rates of crossbred Bos indicus beef cows was evaluated. Pluriparous nursing calv cows (n=120) were synchronized as follows: d 0 cows received a 2.0 mg injection of estradiol benzoate (EB) and insertion of a controlled intravaginal progesterone releasing device containing 0.558 g of progesterone, d 8 removal of the progesterone device , a 0.15 mg injection of prostaglandin F2α (PGF), a 1.0 mg injection of EB, and 400 IU injection of equine chorionic gonadotropin. Fifty-four hr after PGF, all cows were exposed to FTAI and a 0.084 mg injection of GnRH was administered either via Vena caudalis (n=60), or via Longissimus dorsi (n=60). Cows were inseminated with the same sire and by a single AI technician. Pregnancy was determined by the transrectal ultrasonography on d 40 after AI. Cows receiving the intravenous administration of GnRH had higher (P = 0.04) pregnancy rates than the cows receiving the intramuscular injection of GnRH (65 vs 46.6%, respectively). It was concluded that the intravenous administration of GnRH at the time of AI improved the pregnancy rates of crossbred Bos indicus beef cows submitted to FTAI.

Embryo transfer in no cycling Crioula and Quarter horse breeds treated with estradiol cipionate and long-acting progesterone

The aim of this work was to prepare the mares for embryo transfer. In group 1 (G1,treated, n=15), recipient mares in anoestrus or in a transition period were treated with 5.0, 3.0 and 2.0 mg of estradiol cipionate at the days 0, 1 and 2 respectively, beginning at the day of ovulation (DO). From the fourth day on, the mares this group received long-acting progesterone weekly, up to the 120 day of gestation. At D8, the embryo was collected from the donor and transferred to the recipient. At D12, the ultrasonographyc diagnosis of pregnancy was carried out. The control group (G2, not treated, n=20) was formed by cycling recipient mares, displaying ovulation at each 2 to 3 days after the donors mare ovulation. The pregnancy rate was higher (p<0.05) in the mares from G2 (85.0%) than from G1 (53.3%). Thus, it could be concluded that the treated mares although showed lesser pregnancy rate than the cycling mare, were satisfactory alternative to be used mainly when there is no available cycling recipient.

Estrus synchronization and Fixed Time Artificial Insemination (FTAI) in dairy buffaloes during seasonal anestrus

The aim of this work was to study estrus synchronization and fixed time artificial insemination (FTAI) in dairy buffaloes during season anestrus. One hundred thirty-nine dairy buffaloes in seasonal anestrus were divided in two groups as G1(n=66) and G2(n=73). The protocols for both the groups were the same until day (D)14:D0 administration of 2.0 mg estradiol benzoate and implantation of progesterone device (P4) for 14 days; D14 removal of P4 plus 150 mg of cloprostenol and 400 IU of equine chorionic gonadotropin. On D16, G1 received 10 mg of buserelin and G2 100 mg deslorelin acetate. On D17, both the groups were submitted to FTAI. Ultrasonographic examinations of ovaries were performed on D0, D14, D16 and D17. Results showed that pregnancy rates in G1 and G2 were 20 and 41% (p 0.05). Thirty-five percent of the animals in G1 and 54.1% in G2 showed a diameter DF greater than 8.0 mm on D16 (p>0.05). Thus, it could be concluded that the protocols synchronized the estrus, leading the concentration of the parturitions in the period of low milk production. Deslorelin was more efficient than buserelin due the higher percentage of DF ovulation and higher pregnancy rates.

Determination of progesterone concentration during the estrous cycle in dairy cows using a chemiluminescence assay

The study aimed to determine the patterns of serum progesterone concentration in estrous cycle in dairy cows by a chemiluminescence assay (CLIA). Four non-lactating multiparous Jersey cows were used. Animals with a corpus luteum (CL) in any of the ovaries were induced into estrus. Day zero (d0) of the estrous cycle was defined as the day of visible estrus. Blood samples were collected and ultrasonography (US) of the ovaries were performed until a new manifestation of visible estrus was observed. The lengths of the estrous cycles (estrus to estrus) of the four cows were 20, 21, 22, and 23 days. The mean serum concentrations of P4 (x ± s) were 2.8 ± 1.4 ng/mL in proestrus, 2.4 ± 1.5 ng/mL in estrus, 2.0 ± 1.8 ng/mL in metestrus, and 11.9 ± 5.7 ng/mL in diestrus. The follicular and luteal phases of the estrous cycle were established based on P4 concentrations. P4 serum concentrations ≥5.48 ng/mL indicated the presence of functional CL, which was observed from d3 to d12 of the cycle. P4 concentrations decreased from d13 until next estrus. Thus, the previously mentioned P4 serum concentration was established as the limit for a predominantly functional CL. P4 concentrations < 5.48 ng/mL indicated a nonfunctional CL or CL that is not yet fully formed, which is observed in metestrus. A P4 standard curve, constructed based on the Lorentz distribution, was used to determine values of < 5.48 ng/mL of serum P4 concentration for the follicular phase and ≥ 5.48 ng/mL for the luteal phase. Data obtained from US examinations were consistent with P4 concentrations determined using CLIA. In conclusion, the automated CLIA was efficient in determining the P4 concentrations during the various stages of estrous cycles in dairy cows. The findings of the study will help researchers in the animal reproduction field. We recommend the use of CLIA because it´s available in many laboratories worldwide with the ability to process thousands of samples per day.

Sperm subpopulations in ejaculated sperm and spermatozoa recovered from ovine epididymides up to 48 h after death

The objectives of this study were threefold: to identify subpopulations of sperm based on the kinetics of frozen/thawed sheep epididymal spermatozoa or semen collected with an artificial vagina; to evaluate the effects on sperm subpopulations in the thawed samples of post mortem storage at room temperature and the addition of 20% of seminal plasma to the freezing extender and to correlate the percentage of subpopulations with gestation rate following artificial intrauterine insemination. The categorization of the subpopulations was based on sperm kinetic data from Computer Assisted Sperm Analysis (CASA). A hundred ewes were inseminated with thawed spermatozoa and gestation rate was correlated with the proportions of each subpopulation using Pearson correlation matrix and linear regression. Three distinct subpopulations were identified in the thawed samples of either ovine ejaculate collected in artificial vaginas (AV) or ovine spermatozoa retrieved from the cauda epididymis. Subpopulation 1 (SP1) was characterized by spermatozoa with slow and non-linear motion, subpopulation 2 (SP2) was classified as hyperactived spermatozoa and subpopulation 3 (SP3) was composed of spermatozoa with fast, linear motion. The largest subpopulation in all groups was SP1. The semen collected in an artificial vagina had a higher (P<0.05) percentage of SP2 and lower (P<0.05) percentage of SP1 when compared to spermatozoa recovered after death. Increasing time of storage after death had a detrimental effect on sperm samples, increasing (P<0.05) the percentage of SP1 and decreasing (P<0.05) SP2. Length of storage after death was the only variable that influenced, with an inversely proportional relationship, SP3. In samples stored for 48h after death no SP3 spermatozoa were present. The addition of seminal plasma to the cryopreservative decreased (P<0.05) the subpopulation of hyperactived spermatozoa (SP2). We conclude that, after thawing there are three sperm subpopulations in the spermatozoa obtained from the cauda epididymides and the semen collected in AVs and that the relative proportions of these subpopulations varies with the time of storage post mortem and the presence of 20% of seminal plasma in the extender. However, we conclude that these subpopulations do not correlate with fertility after intrauterine artificial insemination.

Impact of Equine Chorionic Gonadotropin Associated with Temporary Weaning, Estradiol Benzoate, or Estradiol Cypionate on Timed Artificial Insemination in Primiparous Bos Indicus Cows

This study aimed to determine the impact of equine chorionic gonadotropin (eCG) associated with different timedartificial insemination (TAI) protocols on the pregnancy rate (PR) in Bos taurus indicus previously treated with progesterone (P4). Five hundred and fifty-seven primiparous cows received the following treatments: on day 0 (d0), GeCGTW (group eCG+Temporary Weaning(TW); n=178) received 0.558g intravaginal progesterone (P4)+1.0 mg of estradiol benzoate (EB)(IM); on d8 (P4 removal+0.075 mg D-cloprostenol+400 IU eCG+TW for 48h); on d10, TAI+calf return to dam; GeCGEB (group eCG+EB; n=176) the same as GeCGTW without TW+1.0 mg of EB on d9; GeCGEC (group eCG+EC;n=203), the same as GeCGTW without TW+1.5 mg estrtadiol cypionate (EC) (IM). On d35 after TAI pregnancy diagnosis (PD) was performed. Open cows remained with clean-up bulls for 90 days and then underwent pregnancy diagnosis by ultrasound. Drugs used were from Biogenesis Bago (Curitiba, PR) except eCG (Foligon, MSD, SP) and the EC (Zoetis, SP). The statistical analysis was performed with Chi-square (level of P 0.05) respectively. The PR following clean-up bulls was 88.3, 47.3, and 31.1% (P<0.05). The final PR (TAI+clean-up bulls) of the groups was 94.4, 72.1, and 64.0%, respectively (P<0.05). In conclusion, there were no detectable differences in PR among TAI protocols; PR in the GeCGTW protocol following clean-up bulls was greater compared to others (P<0.05); the overall PR of cows subjected to TAI+clean-up bulls was significantly greater in GeCGTW as compared to other groups.

Pregnancy and repeat breeding rate of high producing dairy cows for morning, evening or night artificial insemination

The study aimed (1) to verify if artificial insemination (AI), performed in the morning, evening or nighttimehas influence on the pregnancy rate on high production dairy cows, and (2) to determine the repeat breedingrate after the AI at intervals of 01-21, 22-43 and 44-65 days. We analyzed 1980 AI data from HolsteinFriesian cows from years 2004 to 2008. The average milk production on this period was 39 kg/milk/day/cow (305 days of lactation). The AI was performed 12 hours after the first detection of estrus. For the dataanalysis one AI was computed. The AI was done based on conventional method. The incidence of the AI in themorning, evening and night was 29.8; 50.4 and 19.8% respectively; and the pregnancy rate 45.7; 44.9 and48.4%. The frequency of the repeat breeding rate at the interval of 1-21 days on morning, evening and nightafter failed AI was 28.0; 22.3 and 19.8% respectively; at the interval of 22-43 days was 52.0; 52.2 and 55.2%,respectively and at interval of 44-65 days was respectively 20.0; 25.5 and 25.0%. Comparing the three timesof the day each other, the cows showed greater repeat breeding on 22-43 days (P<0.05) after the AI. It wasconcluded that times of the day has no influence on the on pregnancy rate; the highest repeat breeding ratetook place between 22 and 43 days after AI, resulting significant delaying of the open period.

Temporary Calf Removal and Equine Chorionic Gonadotropin (Ecg) Administration after Progesterone-Based Protocol Improves the Reproductive Performance of Beef Cattle

This study aimed to evaluate the effects of equine chorionic gonadotropin (eCG) administration and the temporary 48-h calf removal (CR) in a long-term progesterone (P4)based protocol in suckled Nelore cows. In all, 150 cows were randomly submitted to P4 protocol as follows. At day 0 (d 0), an intravaginal device containing 1.9 g P4 was inserted, and 1 mg estradiol benzoate (EB) was injected intramuscularly (im). On d 8, the P4 device was removed, and cows were im injected 500 µg of prostaglandin F group 2 alpha (PGF 2α ). On the same day, 75 cows (eCGCR group) received 400 IU im of eCG and were separated from their calves for 48 h. The other 75 cows (NoeCGCR group) stayed with the calves and did not receive eCG. On d 10, the calves were returned to the eCGCR group. After 8 days, all the cows were observed for estrus. The ovulatory follicle size was measured using ultrasonography at the day estrus was detected, and artificial insemination (AI) was performed 12 h later. Pregnancy was diagnosed 35 days after AI. The respective parameters for the eCGCR and NoeCGCR groups were as follows: pregnancy rate, 74.6% and 50.6% ( p 0.05). Thus, eCGCR after a long-term P4 protocol markedly improved pregnancy rates and estrus induction.