Fixed-time Insemination Protocol Research Articles

A simplified fixed-time insemination protocol using frozen-thawed stallion spermatozoa stored at 17°C for up to 24 h before insemination.

Insemination of mares with frozen-thawed spermatozoa requires intensive management and results in 40%-60% per cycle pregnancy rates. To determine if satisfactory fertility is possible for frozen-thawed semen after processing it through a microfluidic device, followed by storage at 17°C for up to 24 h before fixed-time insemination. Uncontrolled field trials. A pilot study evaluated the motility of frozen-thawed spermatozoa after centrifugation and storage (17°C) in two different media for up to 48 h. Subsequently, the motility of frozen-thawed semen processed through a microfluidic device, resuspended in two different media during storage (17°C) for up to 24 h was evaluated. The fertility of frozen-thawed spermatozoa, after microfluidic sorting and storage at 17°C for up to 24 h, was evaluated after fixed-time insemination in a commercial embryo programme. Experiment 1: Frozen-thawed spermatozoa (N = 5 stallions) were centrifuged and resuspended in Botusemen Gold™ or SpermSafe™ and stored (17°C) for up to 48 h. Sperm motility was evaluated by CASA at 0, 6, 24 and 48 h. Experiment 2: Frozen-thawed spermatozoa (N = 4 stallions) underwent microfluidic sorting and storage (17°C) for up to 24 h in both media. Sperm concentration and motility were evaluated at 0, 16 and 24 h. Experiment 3: Fertility of frozen-thawed spermatozoa (N = 3 stallions) was evaluated after insemination of 42 mare cycles at 6, 16 and 24 h after thawing, microfluidic sorting and storage before fixed-time insemination. The stallion significantly influenced sperm motility, but there was no effect of media on motility parameters. Storage time significantly affected sperm motility after centrifugation but not after microfluidic sorting. Storage time had no effect on the overall embryo recovery rate (52%, n = 42). Field trial with small mare numbers and no control at time = 0 h. Fixed-time insemination of frozen-thawed spermatozoa after microfluidic sorting and storage at 17°C for up to 24 h produced satisfactory embryo recovery rates.

Clinical prospects proposing an increase in the luteolytic dose of prostaglandin F2α in dairy cattle.

Over the past few decades, the luteolytic dose of prostaglandin F2α (PGF2α) and its analogs, used to synchronize estrus for fixed-time insemination in dairy cattle, have remained unchanged. Given the beneficial effects of PGF2α on a young corpus luteum and on multiple ovulations in a fixed-time insemination protocol, and its therapeutic abortive effects on multiple ovulations in pregnant cows, we propose the use of a double PGF2α dose or two PGF2α treatments 24 hours apart. Ultrasonography procedures serve to identify luteal structures and may therefore help to determine the best PGF2α dose to improve the fertility of high-producing dairy cows.

Benefits of synchronizing ovulation with porcine luteinizing hormone (pLH) in a fixed time insemination protocol in weaned multiparous sows

Materials and methods: Multiparous sows were randomly assigned to treatment at weaning. From weaning, twice-daily boar exposure facilitated estrus detection. Untreated control sows (n = 150; CONT) were artificially inseminated at least twice. Treated sows (n = 168; LUT) received 5 mg pLH intramuscularly concomitant with the first detection of standing heat. To coincide with the normal working day, sows in estrus in the morning were inseminated at 24 and 30 hours (am and pm), while sows in estrus in the afternoon were inseminated at 24 and 42 hours (pm and am), after pLH administration.

Influence of Follicular Growth Stimulation on Fertility of Beef Cows Treated with eCG or FSH in FTAI Protocols

Background: The pregnancy rate in fixed-time insemination (FTAI) programs in beef cattle is affected by several factors, including the size of ovulatory follicle (OF). Induced ovulation of small OF (≤11 mm) reduces the chance of pregnancy after FTAI when compared to ovulation of larger follicles (≥12 mm). Hormonal treatments to enhance the follicle growth have been widely used in comercial beef cattle systems, aiming to improve the development of a dominant follicle. The objective of this experiment was to evaluate the effect of treatment with eCG or FSH at the time of progesterone implant removal on the diameter of OF, final follicle growth and pregnancy rate in beef cows.Materials, Methods & Results: Three hundred and seven suckled beef cows with body condition score 2.80 ± 0.24 (1 to 5 scale) and 45 to 70 days postpartum were synchronized to FTAI. On day 0, intravaginal implants containing 1 g of progesterone, were inserted and administered 2 mg of estradiol benzoate (EB), i.m. On day 7, intravaginal implants were removed and 150 µg of D-cloprostenol were administered i.m. At this time, three groups were assigned according to the treatments: eCG (400 UI eCG, i.m.), FSH (25 UI FSH, i.m.) and Control (1 mL Saline, i.m.). On day 8, all cows were injected with 1mg EB i.m. and were inseminated 52 to 56 h after the implant removal. Transrectal ultrasonography exams of ovaries were performed at the time of implant removal and before FTAI to determine the diameter of the largest follicle and the follicular daily growth. Pregnancy exams were conducted by transretal ultrasonography 30 days after FTAI. Statistical analyzes were performed by Chi-square test for pregnancy rate. Ovarian diameters and follicular daily growth were analyzed by ANOVA with means compared by Tukey test considering significant P value < 0.05. The diameter of the largest follicle measured on day 7 (10.07, 9.64 and 10.36 mm) and on day 9 (12.73, 11.53 and 12.57 mm), to eCG, FSH and Control respectively, did not differ within treatments (P > 0.05). Final follicular growth was greater (P < 0.05) on eCG group (1.33 mm/day) compared to Control (1.1 mm/day) and FSH (0.94 mm/day). The pregnancy rates of FTAI were 56.6% (60/106), 52% (51/98) and 50.4% (52/103) to eCG, FSH and Control, respectively, with no statistic difference (P > 0.05).Discussion: Suckled beef cows managed under grazing systems frequently have reduced follicular development and longer postpartum anestrous. In this situation, protocols to FTAI combining progesterone, estradiol and eCG or FSH at the time of progesterone device removal are encouraged to improve final follicular growth, ovulation and to increase conception rate. The hypotesis of similar follicular growth and pregnancy rate in suckled beef cows treated with eCG or FSH was not confirmed in the field trial. eCG treated cows had greater follicular growth, follicular diameter at day 9 and higher pregnancy rate compared to FSH and Control cows. FSH produced similar follicular growth rates to Controls and both were significantly smaller than observed in cows receiving eCG. Also FSH supplementation failed to enhance pregnancy rate compared to Control cows. The longer half-life of eCG and its greater LH-like effects could explain the difference in final follicular stimulation when compared to FSH in FTAI protocols. Thus, administration of a single dose of FSH at progesterone device removal failed to be an alternative to eCG in FTAI synchronization programs.

Efficacy of Estrus Synchronization Protocols during Summer and Winter Seasons together with Biochemical and Minerals Profile in Anoestrus Crossbred Cows

The estrus induction and fertility response along with biochemical and minerals profile of >90 days postpartum anoestrus crossbred cows following use of different estrus synchronization protocols was investigated during summer and winter seasons. The true anoestrus cows selected (n=43 in summer, n=40 in winter) were randomly treated with Ovsynch, Heatsynch and PRID protocols keeping 10 animals as control in both the seasons. During summer season, the estrus induction response with Ovsynch, Heatsynch and PRID protocol was 80, 90 and 80 %; while in winter season, the response was 100% with all three protocols. In summer season, the conception rates with Ovsynch, Heatsynch and PRID protocols at induced estrus were 30, 20 and 20 %and those of 3 cycles 60, 40 and 50 %, respectively, while in winter season, the conception rates at induced estrus were 45.45, 27.27 and 36.36 % and overall of 3 cycles 72.72, 45.45 and 63.64%, respectively. The overall mean plasma concentrations of total cholesterol (mg/dl) in anoestrus cows under Ovsynch, Heatsynch and PRID protocols were 171.57±4.75, 190.97±4.90 and 184.93±3.10 during winter and 163.73±4.96, 162.87±11.24 and 177.63±7.98 during summer, respectively. The corresponding values for plasma total protein (g/dl) during winter season were 7.77±0.11, 7.90±0.12and7.51±0.14, and in summer season 7.90±0.14, 8.02±0.67 and 8.32±0.10, respectively. The cholesterol levels were insignificantly lower and protein levels were significantly higher in summer than winter season, particularly in non-conceived cows. Among the minerals profiles, i.e. calcium, phosphorus and magnesium, only the calcium levels were significantly higher during winter and in conceived cows as compared to counter parts. In conclusion, the results showed that estrus synchronization with fixed time insemination protocols can augment fertility in anoestrus crossbred cows under favorable as well as heat stress condition, and the response is better with Ovsynch and PRID protocols without influencing much the blood metabolic profil

Recent advancements in the hormonal stimulation of ovulation in swine.

Induction of ovulation for controlled breeding is available for use around the world, and conditions for practical application appear promising. Many of the hormones available, such as human chorionic gonadotropin (hCG), gonadotropin-releasing hormone (GnRH) and its analogs, as well as porcine luteinizing hormone (pLH), have been shown to be effective for advancing or synchronizing ovulation in gilts and weaned sows. Each of the hormones has unique attributes with respect to the physiology of its actions, how it is administered, its efficacy, and approval for use. The timing for induction of ovulation during the follicle phase is critical as follicle maturity changes over time, and the success of the response is determined by the stage of follicle development. Female fertility is also a primary factor affecting the success of ovulation induction and fixed time insemination protocols. Approximately 80%–90% of female pigs will develop mature follicles following weaning in sows and synchronization of estrus in gilts. However, those gilts and sows with follicles that are less developed and mature, or those that develop with abnormalities, will not respond to an ovulatory surge of LH. To address this problem, some protocols induce follicle development in all females, which can improve the overall reliability of the ovulation response. Control of ovulation is practical for use with fixed time artificial insemination and should prove highly advantageous for low-dose and single-service artificial insemination and for use with frozen-thawed and sex-sorted sperm.

Influence of inducing luteal regression before a modified fixed-time artificial insemination protocol in postpartum beef cows on pregnancy success1

Most fixed-time insemination protocols utilize an injection of GnRH at the beginning of the protocol to initiate a new follicular wave. However, the ability of GnRH to initiate a new follicular wave is dependent on the stage of the estrous cycle. We hypothesized that administering PGF(2α) 3 d before initiating a fixed-time AI protocol would improve synchrony of follicular waves and result in greater pregnancy success. Therefore, our objective was to determine whether inducing luteal regression 3 d before a fixed-time AI protocol would improve control of follicular turnover and pregnancy success to fixed-time AI. Multiparous crossbred cows at 3 locations (n = 108, 296, and 97) were randomly assigned to 1 of 2 treatments: 1) PGF(2α) [25 mg; intramuscularly (i.m.)] on d -9, GnRH (100 μg; i.m.) and insertion of a controlled internal drug-releasing device (CIDR) on d -6, PGF(2α) (25 mg; i.m.) and CIDR removal with PGF(2α) (25 mg; i.m.) at CIDR removal on d 0 (PG-CIDR) or 2) GnRH (100 μg; i.m.) and insertion of a CIDR on d -5 and CIDR removal with PGF(2α) (25 mg; i.m.) at CIDR removal and 4 to 6 h after CIDR removal (5-d CIDR). Cows were time-inseminated between 66 and 72 h (PG-CIDR) or 70 to 74 h (5-d CIDR) after CIDR removal, and GnRH was administered at the time of fixed-time AI. At location 1, ovulatory response to the first injection of GnRH was determined by ultrasonography at the time of GnRH and 48 h after GnRH administration. Among cows with follicles ≥10 mm in diameter, more (P = 0.03) PG-CIDR-treated cows ovulated after the initial GnRH injection (88%, 43/49) compared with the 5-d CIDR-treated cows (68%, 34/50). Pregnancy outcome was not influenced by location (P = 0.96), age of the animal (P = 1.0), cycling status (P = 0.99), BCS (P = 1.0), or any 2-way interactions (P ≥ 0.13). However, pregnancy success was influenced by synchronization protocol (P = 0.04). Pregnancy outcome was greater (P = 0.04) for the PG-CIDR protocol (64%) compared with the 5-d CIDR protocol (55%). In summary, control of follicular turnover was improved by inducing luteal regression 3 d before initiation of a fixed-time AI protocol, and pregnancy success was improved with the PG-CIDR protocol compared with the 5-d protocol.

Benefits of synchronizing ovulation with porcine luteinizing hormone in a fixed-time insemination protocol in weaned multiparous sows

Objective: To determine reproductive performance of weaned sows inseminated twice at fixed times after controlling time of ovulation with porcine luteinizing hormone (pLH) administered at onset of behavioral estrus. Materials and methods: Multiparous sows were randomly assigned to treatment at weaning. From weaning, twice-daily boar exposure facilitated estrus detection. Untreated control sows (n = 150; CONT) were artificially inseminated at least twice. Treated sows (n = 168; LUT) received 5 mg pLH intramuscularly concomitant with the first detection of standing heat. To coincide with the normal working day, sows in estrus in the morning were inseminated at 24 and 30 hours (am and pm), while sows in estrus in the afternoon were inseminated at 24 and 42 hours (pm and am), after pLH administration. Results: For multiparous sows bred on days 4 to 6 after weaning, total-born litter size was greater in LUT than in CONT (12.88 and 11.80, respectively; P < .01), whereas adjusted farrowing rate was not affected by treatment, (LUT, 87.28%; CONT, 83.20%; P > .05). Neither variable was affected by day of breeding or a treatment × day interaction (P > .05). Fewer inseminations (P < .001) were performed in LUT (2.00) than in CONT (2.13). Implication: Double insemination of multiparous sows, timed to coincide with optimal sow fertility, may improve litter size.

Strategies to improve fertility in postpartum multiparous Bos indicus cows submitted to a fixed-time insemination protocol with gonadotropin-releasing hormone and prostaglandin F2α1

In Exp. 1, we evaluated the effects of 2 lengths of progesterone exposure [CIDR (controlled intravaginal drug release); 7 vs. 14 d] before a modified CO-Synch protocol [50.0-microg injection of GnRH 6.5 d before a 25.0-mg injection of PGF(2alpha) followed by another injection of GnRH and fixed-time AI (TAI) 2 d after PGF(2alpha)], with or without temporary weaning (TW) before GnRH treatments, on fertility of suckled multiparous Bos indicus cows (n = 283) and on calf performance. Timed AI pregnancy rates for cows receiving 7 d CIDR + TW, 7 d CIDR, 14 d CIDR + TW, and 14 d CIDR were 53, 47, 46, and 41%, respectively (P > 0.10). Calves submitted to two 48-h TW 6 d apart had decreased mean BW at 240 d (187.9 +/- 2.7 vs. 195.5 +/- 2.7 kg; P < 0.05), but BW at 420 d was not affected by TW (240.1 +/- 5.1 kg). In Exp. 2, we evaluated the effect of no treatment and treatment with or without a CIDR insert between GnRH and PGF(2alpha) treatments of a modified CO-Synch protocol on pregnancy rate to TAI, and throughout a 90-d breeding season in suckled multiparous Bos indicus cows (n = 453). The inclusion of a CIDR between first GnRH and PGF(2alpha) treatments of a modified CO-Synch protocol did not improve pregnancy rate (29 and 33% for cows receiving CO-Synch + CIDR and CO-Synch protocol, respectively), and cycling cows had poorer TAI pregnancy rates than anestrous cows treated with either synchronization protocol (21.7 vs. 40.7%; P < 0.05). However, regardless of treatment with CIDR, cows submitted to TAI protocol had greater (P < 0.05) pregnancy rates at 30 (54.8 vs. 11.2%), 60 (72.1 vs. 38.8%), and 90 d (82.0 vs. 57.9%) of breeding season than untreated cows.

A retrospective study of artificial insemination of 251 mares using chilled and fixed time frozen‐thawed semen

Historically, artificial insemination (AI) using frozen semen has been perceived to have poorer success rates and be more labour intensive than using chilled semen. A retrospective study was therefore conducted to compare the conception rate achieved by AI between chilled and frozen semen, using fixed time insemination protocols over 2 breeding seasons. Artificial insemination using chilled semen produces a higher conception rate than that achieved with frozen semen. Mares (n = 251) were inseminated with either chilled (n = 112) or frozen (n = 139) semen in the 2006 and 2007 northern hemisphere breeding season. Per rectum ultrasonography of the mare's reproductive tract determined the timing of insemination, and deslorelin acetate was used to induce ovulation. Chilled semen insemination was performed using a single preovulatory dose delivered into the uterine body. Frozen semen was administered as 2 doses (pre- and post ovulation) using a deep uterine insemination technique. Pregnancy was detected ultrasonographically at 15 days post insemination. Conception rates were compared using a Chi-squared test. Insemination with frozen semen produced a significantly (P = 0.022) higher seasonal conception rate (82.0%) than that achieved with chilled semen (69.6%). Insemination with frozen semen can achieve conception rates equal to those with chilled semen, enabling the mare owner a greater selection of stallions.

Effect of follicle size on fertility in cattle.

Abstract Procedures that control the timing of ovulation are of enormous value in advancing the use of reproductive technologies in all species. Among cattle, most protocols that control the timing of ovulation utilize gonadotrophin-releasing hormone (GnRH) around the time of insemination. A luteinizing hormone (LH) surge and ovulation are induced with an injection of GnRH. However, GnRH results in the ovulation of a wide range of follicle sizes regardless of whether or not standing oestrus had been initiated. Among both heifers and cows, the relationship between follicle size and pregnancy success following a fixed-time insemination protocol was quadratic. Small follicles induced to ovulate resulted in lower pregnancy rates, increased embryonic/ foetal mortality, and a slower rise in subsequent concentrations of progesterone compared with cows induced to ovulate larger follicles. Among postpartum cows, there was no relationship between ovulatory follicle size and pregnancy status when cows spontaneously initiated standing oestrus and ovulated. Furthermore, animals that exhibited standing oestrus within 24 h of fixed-time insemination had greater pregnancy success compared with animals that did not exhibit standing oestrus, and cows that exhibited standing oestrus had greater circulating concentrations of estradiol compared with cows that did not. Therefore, follicular maturity at time of GnRH-induced ovulation can influence pregnancy success following a fixed-time insemination protocol.

Luteinizing hormone release after administration of the gonadotropin-releasing hormone agonist Fertilan (goserelin) for synchronization of ovulation in pigs

The generic GnRH agonist, Fertilan (goserelin), was tested for the ability to induce an LH surge and ovulation in estrus-synchronized gilts. Three experiments were performed to 1) examine the effect of various doses of Fertilan on secretion of LH in barrows, to select doses to investigate in gilts (Exp. 1); 2) determine doses of Fertilan that would induce a preovulatory-like rise of LH in gilts (Exp. 2); and 3) determine the time of ovulation after Fertilan treatment (Exp. 3). In Exp. 1, 10 barrows were injected on d 1, 4, 7, 10, and 13 with 10, 20, or 40 microg of Fertilan; 50 microg of Gonavet (depherelin; GnRH control) or saline (negative control); and sequential blood samples were collected for 480 min. There was a dose-dependent stimulation (P < 0.05) of LH release. Maximal plasma concentrations of LH (LH(MAX)) were 2.1 +/- 0.2, 4.1 +/- 0.3, 2.6 +/- 0.4, and 3.4 +/- 0.3 ng/mL after 10, 20, and 40 microg of Fertilan and 50 microg of Gonavet, respectively, and duration of release was 78 +/- 9, 177 +/- 12, 138 +/- 7, and 180 +/- 11 min, respectively. Fertilan doses of 10 and 20 microg were deemed to be the most suitable for testing in gilts. In Exp. 2, 12 gilts received (after estrus synchronization with Regumate and eCG) injections of 10 or 20 microg of Fertilan or 50 microg of Gonavet 80 h after eCG to stimulate a preovulatory-like LH surge and ovulation. An LH surge was induced in 3 of the 4 gilts in both of the Fertilan groups and in all of the Gonavet-treated gilts. Characteristics of induced release of LH did not differ among groups: LH(MAX), 5.0 +/- 0.9 vs. 4.6 +/- 1.8 vs. 6.6 +/- 1.1 ng/mL; duration, 11.7 +/- 2.0 vs. 12.3 +/- 2.2 vs. 14.3 +/- 0.5 h; interval from GnRH injection to LH(MAX), 4.0 +/- 2.0 vs. 6.7 +/- 1.3 vs. 5.8 +/- 1.6 h. In Exp. 3, estrus-synchronized gilts were injected with 20 microg of Fertilan (n = 8) or 50 microg of Gonavet (n = 4), and the time of ovulation was determined by repeated endoscopic examination. Time of ovulation ranged from 34 to 42 h postGnRH; however, ovulation occurred earlier in the Gonavet compared with the other groups (P < 0.05). Results of these experiments indicate that 1) barrows are an appropriate model for determining GnRH doses that can be effective in inducing a preovulatory-like LH surge in females; 2) the generic GnRH agonist Fertilan, at doses of 10 to 20 microg, can stimulate an LH surge in gilts, with subsequent ovulation; and 3) Fertilan at doses of 10 and 20 microg should be examined further for use in fixed-time insemination protocols.