Buffalo bull spermatozoa are rich in polyunsaturated fatty acids and are prone to lipid peroxidat... more Buffalo bull spermatozoa are rich in polyunsaturated fatty acids and are prone to lipid peroxidation. We hypothesised that lipid peroxidation in buffalo bull semen will increase with age and will affect the semen quality. The objective was to compare malondialdehyde (MDA) concentration and the quality of fresh and frozen-thawed semen between aged (13.6 ± 1.0 years; n ¼ 3) and young (3.4 ± 0.3 years; n ¼ 3) Nili-Ravi bulls. The concentration of MDA did not differ (p >.05) between aged vs. young bulls in fresh (2.3 ± 0.2 vs. 2.9 ± 0.7 nmol mL À1), frozen-thawed (53.1 ± 2.8 vs. 48.4 ± 2.6 nmol mL À1) semen and seminal plasma (5.71 ± 0.97 vs. 5.19 ± 1.36 nmol mL À1), respectively. In fresh semen, sperm motility and total concentration did not differ (p > .05) between aged and young bulls. The volume of fresh semen increased (p > .05) while sperm viability and DNA integrity decreased (p < .05) in aged vs. young bulls. In frozen-thawed semen, sperm motility, viability and DNA integrity decreased (p < .05) in aged vs. young bulls. In frozen-thawed vs. fresh semen, MDA level increased within young (48.4 ± 2.6 vs. 2.3 ± 0.2 nmol mL À1) and aged bulls (53.1 ± 2.8 vs. 2.9 ± 0.7 nmol mL À1). Conversely, sperm motility and viability decreased (p < .05) within the age groups between fresh and frozen-thawed semen. In conclusion, (1) lipid peroxidation (MDA) does not increase due to age; however, it is negatively associated with semen quality and (2) cryopreservation steps up lipid peroxidation irrespective of age and deteriorates semen quality of Nili-Ravi bulls.
This study compares the factors associated with variable interval to estrus and ovulation between... more This study compares the factors associated with variable interval to estrus and ovulation between early vs. late ovulating goats following PGF administration. The extent of ovulation time in Beetal goats (n = 38) was monitored through transrectal ultrasound at every 6 h following a single dose of PGF (experiment 1). Variation in estrus and ovulation times was further explored through the changes in follicular dynamics, endocrine profiles and behavior in another set of goats (n = 13) following single PGF given randomly during the luteal phase (experiment 2). The ovulation time varied between 60 to 96 h and 57% of ovulations occurred by 72 h following PGF (experiment 1). Accordingly, the goats (n = 13) in the second experiment were retrospectively divided either into early and/or late ovulating i.e., ≤ 72 and/or ≥ 84 h following PGF . The onset of estrus, peak estradiol-17β concentration and LH surge after PGF was first observed in early than late ovulating goats (P &amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05). The goats ovulating early had larger follicle and smaller CL in diameter at the time of PGF administration than those ovulating late (5.4 ± 0.2 vs. 4.3 ± 0.2 mm and 10 ± 0.6 vs. 11.8 ± 0.3 mm, respectively; P &amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05). Likewise, plasma progesterone concentration tended to be lower (P = 0.087) in early than late ovulating goats. In conclusion, the size of dominant follicle and CL at the time of PGF determines the interval to ovulation following a single dose of PGF during the luteal phase. This article is protected by copyright. All rights reserved.
The objective was to determine how maternal age influences the transcriptome of the dominant foll... more The objective was to determine how maternal age influences the transcriptome of the dominant follicle during the preovulatory period. We tested the hypotheses that delayed ovulation in aged cows is associated with 1) altered gene expression of granulosa cells of the preovulatory follicle and 2) decreased synthesis of progesterone by granulosa cells of the preovulatory follicle. Granulosa cells of preovulatory follicles were obtained 24 h after LH treatment from aged Hereford cows (17.0 ± 2.5 years; n = 6) and their daughters (9.0 ± 0.6 years; n = 6), and compared using bovine specific microarrays (EMBV3, EmbryoGENE, Québec, QC, Canada). Results were confirmed by RT-qPCR. A total of 1340 genes or gene isoforms were expressed differentially (≥2-fold change; P ≤ 0.05) in aged cows v. their younger daughters. Differentially expressed up- and down-regulated genes were related to 1) LH response (↑RGS2, ↑SERPINE2, ↑PTGS2), 2) cellular differentiation and luteinization (↑TNFAIP6, ↑GADD45B, ...
Relative gene expression analysis using real-time quantitative polymerase chain reaction (RT-qPCR... more Relative gene expression analysis using real-time quantitative polymerase chain reaction (RT-qPCR) requires normalization of target gene expression against reference genes. The stability of the reference genes may vary due to many factors such as physiological status or treatments applied; therefore, selection of reference gene is critical for accurate and reliable gene expression studies. The objective of the study was to determine a set of reference genes in granulosa cells of dominant follicles that are suitable for relative gene expression analyses during maternal and follicular aging. Granulosa cells were collected from growing dominant follicles (Day 3 of follicular wave, n=4) and preovulatory follicles (24h post-LH, n=7) from aged (17±5 years, n=5) and young (8±3 years, n=6) cows (maternal aging study), and from FSH-stimulated follicles (24h post-LH) developing under different durations of FSH treatment (follicular aging study; 4day-FSH, n =3; 7day-FSH, n=2 and FSH-starvation...
In the first experiment, effect of GnRH and PGF 2α given intramuscularly, 9 days apart, was obser... more In the first experiment, effect of GnRH and PGF 2α given intramuscularly, 9 days apart, was observed on induction of ovulation and synchronization of oestrus in anoestrous buffaloes during summer (n=2). Ovarian follicles and corpora lutea were monitored on every other day basis, using B-mode real time, transrectal ultrasonography. Oestrus detection was carried out twice daily. The diameter of the largest follicle on the day of administration of GnRH averaged 9 ± 0 mm. These follicles ovulated within 48 h after injection of GnRH. Interval to oestrus after injection of PGF 2α was 63 ± 11 h. In the second experiment, effect of follicle stimulating hormone (FSH-p) on follicular development in buffaloes during summer (n=2) was observed. FSH-p (total of 40 mg) was administered intramuscularly in divided doses, twice daily on Days 10, 11, 12 and 13. PGF 2α (2 ml) was injected on Day 13. Ovarian follicles and corpora lutea were monitored on daily basis using real time, transrectal ultrasonography. Oestrus detection was carried out twice daily. Superovulatory response was measured and analyzed by comparing follicular development on Day 10, i. e. beginning of FSH-p treatment, (before) and on Day of next oestrus (after). Superovulatory response was confirmed by determining number of corpora lutea on Day 7 after oestrus. Interval to oestrus after injection of PGF 2α was 37 ± 10 h. Mean number of small follicles decreased (P<0.05) 'after' FSH-p treatment than 'before'. Mean number of medium and large follicles and corpora lutea increased (P<0.05) 'after' FSH-p treatment than 'before'. It is concluded that protocol of GnRH-PGF 2α can induce ovulation and oestrus in buffaloes and reasonable superovulatory response to FSH-p can be achieved during summer when given during mid luteal phase.
The review presents an overview of studies that examined the effects of follicular aging and mate... more The review presents an overview of studies that examined the effects of follicular aging and maternal aging in the bovine model. The first of three main sections is a discussion of the developmental competence of oocytes from (1) the ovulatory follicle of 2-wave and 3-wave estrous cycles, (2) dominant follicles that develop under high or low LH pulse frequency, and (3) natural versus FSH-stimulated ovulatory follicles. The second section highlights the effects of maternal aging. Maternal aging in cattle is associated with (1) elevated circulating FSH concentrations, (2) reduced response to superstimulatory treatment, and (3) markedly decreased early embryonic development in cows >12 year of age. The third and final section on superstimulation protocols addresses the effects of the duration of FSH stimulation and withdrawal (i.e., FSH "starvation" or "coasting") on oocyte competence. Ovarian superstimulation for 4 days altered the expression of genes related to...
The aim of the present study was to determine a set of reference genes in granulosa cells of domi... more The aim of the present study was to determine a set of reference genes in granulosa cells of dominant follicles that are suitable for relative gene expression analyses during maternal and follicular aging. Granulosa cells of growing and preovulatory dominant follicles were collected from aged and young cows (maternal aging study) and from FSH-stimulated follicles developing under different durations of FSH treatment (follicular aging study). The mRNA levels of the two commonly used reference genes (GAPDH, ACTB) and four novel genes (UBE2D2, EIF2B2, SF3A1, RNF20) were analysed using cycle threshold values. Results revealed that mRNA levels of GAPDH, ACTB, EIF2B2, RNF20, SF3A1 and UBE2D2 were similar (P > 0.05) between dominant follicle type, age and among follicles obtained after FSH-stimulation, but differed (P = 0.005) due to mRNA processing (i.e. with versus without amplification). The stability of reference genes was analysed using GeNorm, DeltaCT and NormFinder programs and c...
Growing dominant follicles at the time of selection from aged (n = 3; 15 ± 1.5 years) and young (... more Growing dominant follicles at the time of selection from aged (n = 3; 15 ± 1.5 years) and young (n = 3; 6 ± 1.1 years) Hereford cows were compared using bovine-specific microarrays containing 40 000 targets. The objective of the study was to determine age-associated changes in transcriptome of granulosa cells at the time of dominant follicle selection. Cows were given prostaglandin F2α to cause ovulation (Day 0) and granulosa cells from dominant follicles were collected on Day 3 either by ultrasound-guided follicle aspiration or after ovariectomy. The mRNA was extracted, analyzed for quality, converted into antisense RNA, amplified, labelled with red and green florescent dyes, and hybridized with microarrays. Feature intensities were measured using Array-Pro software (Media Cybernetics Inc., Rockville, MD), and differentially expressed genes were obtained using FlexArray 1.6. A total of 169 transcripts were differentially expressed with a fold change of ≥2 (P ≤ 0.05) in aged cows v....
Microarray analysis was used to compare the gene expression of granulosa cells from dominant foll... more Microarray analysis was used to compare the gene expression of granulosa cells from dominant follicles with that of those after superstimulatory treatment. Cows were allocated randomly to two groups (superstimulation and control, n=6/group). A new follicular wave was induced by ablation of follicles ≥5 mm in diameter, and a progesterone-releasing device controlled internal drug release (CIDR) was placed in the vagina. The superstimulation group was given eight doses of 25 mg FSH at 12-h intervals starting from the day of wave emergence (day 0), whereas the control group was not given FSH treatment. Both groups were given prostaglandin F2α twice, 12 h apart, on day 3 and the CIDR was removed at the second injection; 25 mg porcine luteinizing hormone (pLH) was given 24 h after CIDR removal, and cows were ovariectomized 24 h later. Granulosa cells were collected for RNA extraction, amplification, and microarray hybridization. A total of 190 genes were downregulated and 280 genes were u...
Hypo-osmotic swelling test (HOST), eosin-nigrosin staining and normal apical ridge test (NAR) wer... more Hypo-osmotic swelling test (HOST), eosin-nigrosin staining and normal apical ridge test (NAR) were used to determine integrity of plasma membrane and acrosome of undiluted, diluted (cooled to 5 o C) and frozen-thawed sperm. Semen from seven bulls was used. For diluted and frozen-thawed sperm, three doses were pooled at 37 o C. Percentage motility was assessed using a phase contrast microscope. A 50µl each of undiluted, diluted and frozen-thawed semen was mixed with 500µl of 50, 100, 150, 150, 190 or 250 mOsm hypo-osmotic treatments of sodium citrate plus fructose and incubated at 37 o C for 1 h. Total number of intact sperm (live) of undiluted, diluted and frozen-thawed were assessed before HOST. Percentage motility decreased (P<0.05) among undiluted (81.0 ± 1.57), diluted (69.6 ± 2.24) and frozen thawed (60.1 ± 1.34) sperm. Swellings of plasma membrane of diluted sperm were higher (P<0.05) at 50 and 100 mOsm than undiluted sperm. Similarly, swellings of diluted sperm were higher (P<0.05) than frozen-thawed sperm. Swellings of undiluted sperm were higher (P<0.05) at 100, 150, 190 and 250 mOsm than frozenthawed sperm. Live sperm were higher (P<0.05) in undiluted (174.4 ± 7.33) and diluted (175.6 ± 3.76) as compared to frozen-thawed (142.3 ± 4.84) semen. Integrity of acrosome in undiluted, diluted and frozen-thawed sperm did not differ (P>0.05), but varied significantly (P<0.05) within bulls. In conclusion, plasma membrane integrity of undiluted and diluted sperm was compromised during freezing and thawing. However, freezing had no effect on acrosomal integrity.
Growing dominant follicles at the time of selection from aged (n = 3; 15 ± 1.5 years) and young (... more Growing dominant follicles at the time of selection from aged (n = 3; 15 ± 1.5 years) and young (n = 3; 6 ± 1.1 years) Hereford cows were compared using bovine-specific microarrays containing 40 000 targets. The objective of the study was to determine age-associated changes in transcriptome of granulosa cells at the time of dominant follicle selection. Cows were given prostaglandin F to cause ovulation (Day 0) and granulosa cells from dominant follicles were collected on Day 3 either by ultrasound-guided follicle aspiration or after ovariectomy. The mRNA was extracted, analyzed for quality, converted into antisense RNA, amplified, labelled with red and green florescent dyes, and hybridized with microarrays. Feature intensities were measured using Array-Pro software (Media Cybernetics Inc., Rockville, MD), and differentially expressed genes were obtained using FlexArray 1.6. A total of 169 transcripts were differentially expressed with a fold change of ≥2 (P ≤ 0.05) in aged cows v. young cows. Ingenuity System Pathway (IPA;
Hypo-osmotic swelling test (HOST), eosin-nigrosin staining and normal apical ridge test (NAR) wer... more Hypo-osmotic swelling test (HOST), eosin-nigrosin staining and normal apical ridge test (NAR) were used to determine integrity of plasma membrane and acrosome of undiluted, diluted (cooled to 5 o C) and frozen-thawed sperm. Semen from seven bulls was used. For diluted and frozen-thawed sperm, three doses were pooled at 37 o C. Percentage motility was assessed using a phase contrast microscope. A 50µl each of undiluted, diluted and frozen-thawed semen was mixed with 500µl of 50, 100, 150, 150, 190 or 250 mOsm hypo-osmotic treatments of sodium citrate plus fructose and incubated at 37 o C for 1 h. Total number of intact sperm (live) of undiluted, diluted and frozen-thawed were assessed before HOST. Percentage motility decreased (P<0.05) among undiluted (81.0 ± 1.57), diluted (69.6 ± 2.24) and frozen thawed (60.1 ± 1.34) sperm. Swellings of plasma membrane of diluted sperm were higher (P<0.05) at 50 and 100 mOsm than undiluted sperm. Similarly, swellings of diluted sperm were higher (P<0.05) than frozen-thawed sperm. Swellings of undiluted sperm were higher (P<0.05) at 100, 150, 190 and 250 mOsm than frozenthawed sperm. Live sperm were higher (P<0.05) in undiluted (174.4 ± 7.33) and diluted (175.6 ± 3.76) as compared to frozen-thawed (142.3 ± 4.84) semen. Integrity of acrosome in undiluted, diluted and frozen-thawed sperm did not differ (P>0.05), but varied significantly (P<0.05) within bulls. In conclusion, plasma membrane integrity of undiluted and diluted sperm was compromised during freezing and thawing. However, freezing had no effect on acrosomal integrity.
In the first experiment, osmotic pressure of semen and seminal plasma in a semen sample from each... more In the first experiment, osmotic pressure of semen and seminal plasma in a semen sample from each of the 20 mature Nili-Ravi buffalo bulls was determined. In the second experiment, effects of osmotic pressure on motility (%), plasma membrane integrity (%) and viability (%) in fresh and frozen-thawed semen samples from each of the seven mature Nili-Ravi buffalo bulls was determined. In the first experiment, seminal plasma was harvested by centrifuging semen at 400 × g for 10 min at 37°C and osmotic pressure was determined using an osmometer. In the second experiment, motility (%) was assessed in fresh and frozen-thawed (37°C for 30 s) semen samples using a phase-contrast microscope (×400). Plasma membrane integrity (%) was determined by mixing 50 μl each of fresh and frozen-thawed semen with 500 μl of solution having an osmotic pressure of 50, 100, 150, 190 or 250 mOsm/l (hypotonic treatments of fructose + sodium citrate) and incubating at 37°C for 1 h. Viability (%) of fresh and fro...
The objective of this study was to determine the effect of GnRH and estradiol benzoate (EB) on fo... more The objective of this study was to determine the effect of GnRH and estradiol benzoate (EB) on follicular wave emergence, estrus, ovulation and pregnancy rate in CIDR treated buffaloes. Buffaloes were randomly selected into three treatment groups which received either GnRH, (100 μg; Dalmarelin; Fatro, Italy; 2 ml, i.m.); (n = 9) or EB (3 mg plus 50 mg progesterone; Duoton Fort; T.P Drug, Lab., Thailand; 2 ml; i.m.); (n = 6) along with insertion of CIDR (day 0) while buffaloes (n = 10) with CIDR alone served as control (CON). All Buffaloes were administered PGF 2α , (0.150 mg; Dalmazine, Fatro, Italy; 2 ml; i.m.) on day 6 followed by removal of CIDR on day 7. Follicular development and ovulation were monitored daily from day 0 until ovulation using real time B-mode ultrasound (Honda; Model: HS-1500; 7.0 MHz). Estrus detection was carried twice daily using penile deviated teaser bull. Buffaloes were once inseminated 12 h after the standing oestrus. Pregnancy diagnosis was carried out on day 45 post insemination using ultrasonography. The results showed that the follicular wave emergence did not differ due to treatments (P > 0.05); however, wave emerged was earlier in buffaloes given GnRH and EB (2.8 ± 0.5 day) than control (4.0 ± 0.5 day). Similarly, the proportion of buffaloes showing estrus did not differ (P > 0.05) among the groups. The interval from PGF 2α administration to estrus (64 ± 12.3 h) did not vary due to treatments but was numerically longer in buffaloes given GnRH. The interval to ovulation after estrus (38.6 ± 7.9 h) did not change because of the treatments. There was no significant difference in ovualtory follicle diameter (9.9 ± 0.5 mm) and pregnancy rate (6/19, 30 %) across the treatments. In conclusion, this preliminary data indicate that administration of GnRH and EB result in emergence of follicular wave, estrus and ovulation and similar pregnancy rate in CIDR treated buffaloes.
The objective of this study was to determine the effect of in utero and lactational exposure to n... more The objective of this study was to determine the effect of in utero and lactational exposure to nicotine on the male reproductive tract. Dams were randomly assigned to receive saline or nicotine bitartrate (1mg/kg-d s.c.) daily for two weeks prior to mating until weaning (postnatal day 21). Male offspring were sacrificed at 7 (peri-pubertal) and 26 (adult) weeks of age.
Buffalo bull spermatozoa are rich in polyunsaturated fatty acids and are prone to lipid peroxidat... more Buffalo bull spermatozoa are rich in polyunsaturated fatty acids and are prone to lipid peroxidation. We hypothesised that lipid peroxidation in buffalo bull semen will increase with age and will affect the semen quality. The objective was to compare malondialdehyde (MDA) concentration and the quality of fresh and frozen-thawed semen between aged (13.6 ± 1.0 years; n ¼ 3) and young (3.4 ± 0.3 years; n ¼ 3) Nili-Ravi bulls. The concentration of MDA did not differ (p >.05) between aged vs. young bulls in fresh (2.3 ± 0.2 vs. 2.9 ± 0.7 nmol mL À1), frozen-thawed (53.1 ± 2.8 vs. 48.4 ± 2.6 nmol mL À1) semen and seminal plasma (5.71 ± 0.97 vs. 5.19 ± 1.36 nmol mL À1), respectively. In fresh semen, sperm motility and total concentration did not differ (p > .05) between aged and young bulls. The volume of fresh semen increased (p > .05) while sperm viability and DNA integrity decreased (p < .05) in aged vs. young bulls. In frozen-thawed semen, sperm motility, viability and DNA integrity decreased (p < .05) in aged vs. young bulls. In frozen-thawed vs. fresh semen, MDA level increased within young (48.4 ± 2.6 vs. 2.3 ± 0.2 nmol mL À1) and aged bulls (53.1 ± 2.8 vs. 2.9 ± 0.7 nmol mL À1). Conversely, sperm motility and viability decreased (p < .05) within the age groups between fresh and frozen-thawed semen. In conclusion, (1) lipid peroxidation (MDA) does not increase due to age; however, it is negatively associated with semen quality and (2) cryopreservation steps up lipid peroxidation irrespective of age and deteriorates semen quality of Nili-Ravi bulls.
This study compares the factors associated with variable interval to estrus and ovulation between... more This study compares the factors associated with variable interval to estrus and ovulation between early vs. late ovulating goats following PGF administration. The extent of ovulation time in Beetal goats (n = 38) was monitored through transrectal ultrasound at every 6 h following a single dose of PGF (experiment 1). Variation in estrus and ovulation times was further explored through the changes in follicular dynamics, endocrine profiles and behavior in another set of goats (n = 13) following single PGF given randomly during the luteal phase (experiment 2). The ovulation time varied between 60 to 96 h and 57% of ovulations occurred by 72 h following PGF (experiment 1). Accordingly, the goats (n = 13) in the second experiment were retrospectively divided either into early and/or late ovulating i.e., ≤ 72 and/or ≥ 84 h following PGF . The onset of estrus, peak estradiol-17β concentration and LH surge after PGF was first observed in early than late ovulating goats (P &amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05). The goats ovulating early had larger follicle and smaller CL in diameter at the time of PGF administration than those ovulating late (5.4 ± 0.2 vs. 4.3 ± 0.2 mm and 10 ± 0.6 vs. 11.8 ± 0.3 mm, respectively; P &amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05). Likewise, plasma progesterone concentration tended to be lower (P = 0.087) in early than late ovulating goats. In conclusion, the size of dominant follicle and CL at the time of PGF determines the interval to ovulation following a single dose of PGF during the luteal phase. This article is protected by copyright. All rights reserved.
The objective was to determine how maternal age influences the transcriptome of the dominant foll... more The objective was to determine how maternal age influences the transcriptome of the dominant follicle during the preovulatory period. We tested the hypotheses that delayed ovulation in aged cows is associated with 1) altered gene expression of granulosa cells of the preovulatory follicle and 2) decreased synthesis of progesterone by granulosa cells of the preovulatory follicle. Granulosa cells of preovulatory follicles were obtained 24 h after LH treatment from aged Hereford cows (17.0 ± 2.5 years; n = 6) and their daughters (9.0 ± 0.6 years; n = 6), and compared using bovine specific microarrays (EMBV3, EmbryoGENE, Québec, QC, Canada). Results were confirmed by RT-qPCR. A total of 1340 genes or gene isoforms were expressed differentially (≥2-fold change; P ≤ 0.05) in aged cows v. their younger daughters. Differentially expressed up- and down-regulated genes were related to 1) LH response (↑RGS2, ↑SERPINE2, ↑PTGS2), 2) cellular differentiation and luteinization (↑TNFAIP6, ↑GADD45B, ...
Relative gene expression analysis using real-time quantitative polymerase chain reaction (RT-qPCR... more Relative gene expression analysis using real-time quantitative polymerase chain reaction (RT-qPCR) requires normalization of target gene expression against reference genes. The stability of the reference genes may vary due to many factors such as physiological status or treatments applied; therefore, selection of reference gene is critical for accurate and reliable gene expression studies. The objective of the study was to determine a set of reference genes in granulosa cells of dominant follicles that are suitable for relative gene expression analyses during maternal and follicular aging. Granulosa cells were collected from growing dominant follicles (Day 3 of follicular wave, n=4) and preovulatory follicles (24h post-LH, n=7) from aged (17±5 years, n=5) and young (8±3 years, n=6) cows (maternal aging study), and from FSH-stimulated follicles (24h post-LH) developing under different durations of FSH treatment (follicular aging study; 4day-FSH, n =3; 7day-FSH, n=2 and FSH-starvation...
In the first experiment, effect of GnRH and PGF 2α given intramuscularly, 9 days apart, was obser... more In the first experiment, effect of GnRH and PGF 2α given intramuscularly, 9 days apart, was observed on induction of ovulation and synchronization of oestrus in anoestrous buffaloes during summer (n=2). Ovarian follicles and corpora lutea were monitored on every other day basis, using B-mode real time, transrectal ultrasonography. Oestrus detection was carried out twice daily. The diameter of the largest follicle on the day of administration of GnRH averaged 9 ± 0 mm. These follicles ovulated within 48 h after injection of GnRH. Interval to oestrus after injection of PGF 2α was 63 ± 11 h. In the second experiment, effect of follicle stimulating hormone (FSH-p) on follicular development in buffaloes during summer (n=2) was observed. FSH-p (total of 40 mg) was administered intramuscularly in divided doses, twice daily on Days 10, 11, 12 and 13. PGF 2α (2 ml) was injected on Day 13. Ovarian follicles and corpora lutea were monitored on daily basis using real time, transrectal ultrasonography. Oestrus detection was carried out twice daily. Superovulatory response was measured and analyzed by comparing follicular development on Day 10, i. e. beginning of FSH-p treatment, (before) and on Day of next oestrus (after). Superovulatory response was confirmed by determining number of corpora lutea on Day 7 after oestrus. Interval to oestrus after injection of PGF 2α was 37 ± 10 h. Mean number of small follicles decreased (P<0.05) 'after' FSH-p treatment than 'before'. Mean number of medium and large follicles and corpora lutea increased (P<0.05) 'after' FSH-p treatment than 'before'. It is concluded that protocol of GnRH-PGF 2α can induce ovulation and oestrus in buffaloes and reasonable superovulatory response to FSH-p can be achieved during summer when given during mid luteal phase.
The review presents an overview of studies that examined the effects of follicular aging and mate... more The review presents an overview of studies that examined the effects of follicular aging and maternal aging in the bovine model. The first of three main sections is a discussion of the developmental competence of oocytes from (1) the ovulatory follicle of 2-wave and 3-wave estrous cycles, (2) dominant follicles that develop under high or low LH pulse frequency, and (3) natural versus FSH-stimulated ovulatory follicles. The second section highlights the effects of maternal aging. Maternal aging in cattle is associated with (1) elevated circulating FSH concentrations, (2) reduced response to superstimulatory treatment, and (3) markedly decreased early embryonic development in cows >12 year of age. The third and final section on superstimulation protocols addresses the effects of the duration of FSH stimulation and withdrawal (i.e., FSH "starvation" or "coasting") on oocyte competence. Ovarian superstimulation for 4 days altered the expression of genes related to...
The aim of the present study was to determine a set of reference genes in granulosa cells of domi... more The aim of the present study was to determine a set of reference genes in granulosa cells of dominant follicles that are suitable for relative gene expression analyses during maternal and follicular aging. Granulosa cells of growing and preovulatory dominant follicles were collected from aged and young cows (maternal aging study) and from FSH-stimulated follicles developing under different durations of FSH treatment (follicular aging study). The mRNA levels of the two commonly used reference genes (GAPDH, ACTB) and four novel genes (UBE2D2, EIF2B2, SF3A1, RNF20) were analysed using cycle threshold values. Results revealed that mRNA levels of GAPDH, ACTB, EIF2B2, RNF20, SF3A1 and UBE2D2 were similar (P > 0.05) between dominant follicle type, age and among follicles obtained after FSH-stimulation, but differed (P = 0.005) due to mRNA processing (i.e. with versus without amplification). The stability of reference genes was analysed using GeNorm, DeltaCT and NormFinder programs and c...
Growing dominant follicles at the time of selection from aged (n = 3; 15 ± 1.5 years) and young (... more Growing dominant follicles at the time of selection from aged (n = 3; 15 ± 1.5 years) and young (n = 3; 6 ± 1.1 years) Hereford cows were compared using bovine-specific microarrays containing 40 000 targets. The objective of the study was to determine age-associated changes in transcriptome of granulosa cells at the time of dominant follicle selection. Cows were given prostaglandin F2α to cause ovulation (Day 0) and granulosa cells from dominant follicles were collected on Day 3 either by ultrasound-guided follicle aspiration or after ovariectomy. The mRNA was extracted, analyzed for quality, converted into antisense RNA, amplified, labelled with red and green florescent dyes, and hybridized with microarrays. Feature intensities were measured using Array-Pro software (Media Cybernetics Inc., Rockville, MD), and differentially expressed genes were obtained using FlexArray 1.6. A total of 169 transcripts were differentially expressed with a fold change of ≥2 (P ≤ 0.05) in aged cows v....
Microarray analysis was used to compare the gene expression of granulosa cells from dominant foll... more Microarray analysis was used to compare the gene expression of granulosa cells from dominant follicles with that of those after superstimulatory treatment. Cows were allocated randomly to two groups (superstimulation and control, n=6/group). A new follicular wave was induced by ablation of follicles ≥5 mm in diameter, and a progesterone-releasing device controlled internal drug release (CIDR) was placed in the vagina. The superstimulation group was given eight doses of 25 mg FSH at 12-h intervals starting from the day of wave emergence (day 0), whereas the control group was not given FSH treatment. Both groups were given prostaglandin F2α twice, 12 h apart, on day 3 and the CIDR was removed at the second injection; 25 mg porcine luteinizing hormone (pLH) was given 24 h after CIDR removal, and cows were ovariectomized 24 h later. Granulosa cells were collected for RNA extraction, amplification, and microarray hybridization. A total of 190 genes were downregulated and 280 genes were u...
Hypo-osmotic swelling test (HOST), eosin-nigrosin staining and normal apical ridge test (NAR) wer... more Hypo-osmotic swelling test (HOST), eosin-nigrosin staining and normal apical ridge test (NAR) were used to determine integrity of plasma membrane and acrosome of undiluted, diluted (cooled to 5 o C) and frozen-thawed sperm. Semen from seven bulls was used. For diluted and frozen-thawed sperm, three doses were pooled at 37 o C. Percentage motility was assessed using a phase contrast microscope. A 50µl each of undiluted, diluted and frozen-thawed semen was mixed with 500µl of 50, 100, 150, 150, 190 or 250 mOsm hypo-osmotic treatments of sodium citrate plus fructose and incubated at 37 o C for 1 h. Total number of intact sperm (live) of undiluted, diluted and frozen-thawed were assessed before HOST. Percentage motility decreased (P<0.05) among undiluted (81.0 ± 1.57), diluted (69.6 ± 2.24) and frozen thawed (60.1 ± 1.34) sperm. Swellings of plasma membrane of diluted sperm were higher (P<0.05) at 50 and 100 mOsm than undiluted sperm. Similarly, swellings of diluted sperm were higher (P<0.05) than frozen-thawed sperm. Swellings of undiluted sperm were higher (P<0.05) at 100, 150, 190 and 250 mOsm than frozenthawed sperm. Live sperm were higher (P<0.05) in undiluted (174.4 ± 7.33) and diluted (175.6 ± 3.76) as compared to frozen-thawed (142.3 ± 4.84) semen. Integrity of acrosome in undiluted, diluted and frozen-thawed sperm did not differ (P>0.05), but varied significantly (P<0.05) within bulls. In conclusion, plasma membrane integrity of undiluted and diluted sperm was compromised during freezing and thawing. However, freezing had no effect on acrosomal integrity.
Growing dominant follicles at the time of selection from aged (n = 3; 15 ± 1.5 years) and young (... more Growing dominant follicles at the time of selection from aged (n = 3; 15 ± 1.5 years) and young (n = 3; 6 ± 1.1 years) Hereford cows were compared using bovine-specific microarrays containing 40 000 targets. The objective of the study was to determine age-associated changes in transcriptome of granulosa cells at the time of dominant follicle selection. Cows were given prostaglandin F to cause ovulation (Day 0) and granulosa cells from dominant follicles were collected on Day 3 either by ultrasound-guided follicle aspiration or after ovariectomy. The mRNA was extracted, analyzed for quality, converted into antisense RNA, amplified, labelled with red and green florescent dyes, and hybridized with microarrays. Feature intensities were measured using Array-Pro software (Media Cybernetics Inc., Rockville, MD), and differentially expressed genes were obtained using FlexArray 1.6. A total of 169 transcripts were differentially expressed with a fold change of ≥2 (P ≤ 0.05) in aged cows v. young cows. Ingenuity System Pathway (IPA;
Hypo-osmotic swelling test (HOST), eosin-nigrosin staining and normal apical ridge test (NAR) wer... more Hypo-osmotic swelling test (HOST), eosin-nigrosin staining and normal apical ridge test (NAR) were used to determine integrity of plasma membrane and acrosome of undiluted, diluted (cooled to 5 o C) and frozen-thawed sperm. Semen from seven bulls was used. For diluted and frozen-thawed sperm, three doses were pooled at 37 o C. Percentage motility was assessed using a phase contrast microscope. A 50µl each of undiluted, diluted and frozen-thawed semen was mixed with 500µl of 50, 100, 150, 150, 190 or 250 mOsm hypo-osmotic treatments of sodium citrate plus fructose and incubated at 37 o C for 1 h. Total number of intact sperm (live) of undiluted, diluted and frozen-thawed were assessed before HOST. Percentage motility decreased (P<0.05) among undiluted (81.0 ± 1.57), diluted (69.6 ± 2.24) and frozen thawed (60.1 ± 1.34) sperm. Swellings of plasma membrane of diluted sperm were higher (P<0.05) at 50 and 100 mOsm than undiluted sperm. Similarly, swellings of diluted sperm were higher (P<0.05) than frozen-thawed sperm. Swellings of undiluted sperm were higher (P<0.05) at 100, 150, 190 and 250 mOsm than frozenthawed sperm. Live sperm were higher (P<0.05) in undiluted (174.4 ± 7.33) and diluted (175.6 ± 3.76) as compared to frozen-thawed (142.3 ± 4.84) semen. Integrity of acrosome in undiluted, diluted and frozen-thawed sperm did not differ (P>0.05), but varied significantly (P<0.05) within bulls. In conclusion, plasma membrane integrity of undiluted and diluted sperm was compromised during freezing and thawing. However, freezing had no effect on acrosomal integrity.
In the first experiment, osmotic pressure of semen and seminal plasma in a semen sample from each... more In the first experiment, osmotic pressure of semen and seminal plasma in a semen sample from each of the 20 mature Nili-Ravi buffalo bulls was determined. In the second experiment, effects of osmotic pressure on motility (%), plasma membrane integrity (%) and viability (%) in fresh and frozen-thawed semen samples from each of the seven mature Nili-Ravi buffalo bulls was determined. In the first experiment, seminal plasma was harvested by centrifuging semen at 400 × g for 10 min at 37°C and osmotic pressure was determined using an osmometer. In the second experiment, motility (%) was assessed in fresh and frozen-thawed (37°C for 30 s) semen samples using a phase-contrast microscope (×400). Plasma membrane integrity (%) was determined by mixing 50 μl each of fresh and frozen-thawed semen with 500 μl of solution having an osmotic pressure of 50, 100, 150, 190 or 250 mOsm/l (hypotonic treatments of fructose + sodium citrate) and incubating at 37°C for 1 h. Viability (%) of fresh and fro...
The objective of this study was to determine the effect of GnRH and estradiol benzoate (EB) on fo... more The objective of this study was to determine the effect of GnRH and estradiol benzoate (EB) on follicular wave emergence, estrus, ovulation and pregnancy rate in CIDR treated buffaloes. Buffaloes were randomly selected into three treatment groups which received either GnRH, (100 μg; Dalmarelin; Fatro, Italy; 2 ml, i.m.); (n = 9) or EB (3 mg plus 50 mg progesterone; Duoton Fort; T.P Drug, Lab., Thailand; 2 ml; i.m.); (n = 6) along with insertion of CIDR (day 0) while buffaloes (n = 10) with CIDR alone served as control (CON). All Buffaloes were administered PGF 2α , (0.150 mg; Dalmazine, Fatro, Italy; 2 ml; i.m.) on day 6 followed by removal of CIDR on day 7. Follicular development and ovulation were monitored daily from day 0 until ovulation using real time B-mode ultrasound (Honda; Model: HS-1500; 7.0 MHz). Estrus detection was carried twice daily using penile deviated teaser bull. Buffaloes were once inseminated 12 h after the standing oestrus. Pregnancy diagnosis was carried out on day 45 post insemination using ultrasonography. The results showed that the follicular wave emergence did not differ due to treatments (P > 0.05); however, wave emerged was earlier in buffaloes given GnRH and EB (2.8 ± 0.5 day) than control (4.0 ± 0.5 day). Similarly, the proportion of buffaloes showing estrus did not differ (P > 0.05) among the groups. The interval from PGF 2α administration to estrus (64 ± 12.3 h) did not vary due to treatments but was numerically longer in buffaloes given GnRH. The interval to ovulation after estrus (38.6 ± 7.9 h) did not change because of the treatments. There was no significant difference in ovualtory follicle diameter (9.9 ± 0.5 mm) and pregnancy rate (6/19, 30 %) across the treatments. In conclusion, this preliminary data indicate that administration of GnRH and EB result in emergence of follicular wave, estrus and ovulation and similar pregnancy rate in CIDR treated buffaloes.
The objective of this study was to determine the effect of in utero and lactational exposure to n... more The objective of this study was to determine the effect of in utero and lactational exposure to nicotine on the male reproductive tract. Dams were randomly assigned to receive saline or nicotine bitartrate (1mg/kg-d s.c.) daily for two weeks prior to mating until weaning (postnatal day 21). Male offspring were sacrificed at 7 (peri-pubertal) and 26 (adult) weeks of age.
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Papers by Muhammad Irfan-ur- Rehman Khan