W. Van Wettere

ABSTRACT A reliable in vitro system for the production of porcine embryos (IVP) is important for use in basic research and in the reproductive technologies. Despite recent developments, blastocyst rates remain low compared with those obtained for most domestic species. Here, we report a porcine IVP system, based largely on our ovine and bovine systems (Walker et al. 1996 Biol. Reprod. 55, 703-708; Kelly et al. 2007 Reprod. Dom. Anim. 42, 577-582), that gives blastocyst development rates higher than previously reported. Abattoir-sourced ovaries were collected into PBS, and cumulus-oocyte complexes (COC) were aspirated (from 3- to 6-mm follicles) into HEPES-TCM-199 supplemented with 0.4% BSA. The COC (up to 30/well) were matured in 600μL of maturation medium (TCM-199, 20% (vol/vol) porcine follicular fluid, FSH, LH, epidermal growth factor, cysteamine, and estradiol) for 42h at 38.6°C in humidified 5% CO(2). After 42h, excess cumulus cells were removed and the COC were placed into modified Tris medium supplemented with BSA and caffeine (500μLwell(-1)). Sperm from pooled semen were washed in HEPES SOF supplemented with BSA, caffeine, and heparin and incubated for 45min. Sperm were then centrifuged and the pellet was resuspended; a concentration of 5×10(5) spermmL(-1) was used. Oocytes and sperm were co-cultured for 6h, cumulus cells were removed, and presumptive zygotes were placed into 600μL of culture medium (SOF, BSA, and amino acids). Zygotes were cultured in 5% CO(2):5% O(2):90% N(2), and cleavage and embryo development to Day 6 were assessed. Separate studies were conducted with COC from mature sows and from prepubertal gilts. In the latter, COC were exposed to ±dbcAMP for the first 22h of the maturation period. Blastocyst rates in the sow were similar to those routinely produced in sheep and cows in our laboratory. Cleavage rates, mean blastocyst cell numbers, and incidence of polyspermy (assessed to be 10 to 12% in prepubertal gilts) were improvements on those generally reported (see Nagai et al. 2006 Front. Biosci. 11, 2565-2573 and Gil et al. 2010 Reprod. Dom. Anim. 45, 40-48 for general reviews). Observations indicate that the use of dbcAMP to regulate oocyte maturation had a negative effect on most parameters. Although blastocyst cell numbers are indicative of blastocyst quality, the significance of these findings can be validated only by transfer studies to determine embryo viability.

ABSTRACT The efficacy of 400 IU equine chorionic gonadotrophin (eCG) and 200 IU human chorionic gonadotrophin (hCG) (PG600; Intervet, Australia) and boar exposure as stimuli of oestrus and ovulation have been established in pre-pubertal gilts and weaned sows, but there is little published data on their use to overcome lactation anoestrus. Designed as a 2 x 2 factorial, the current study used 40 Large White/Landrace sows (parity 1.9 +/- 0.14), to determine the effects of boar exposure (BE) versus NoBE and PG600 versus NoPG600 on the incidence of lactation oestrus and ovulation. On day 1 of lactation, PG600 was administered and BE commenced. Throughout lactation all sows were housed individually in farrowing crates (0.6 x 2.4 m(2)). BE consisted of 15 min of daily, full physical boar exposure in a detection mating area. For all sows, litter size suckled was standardized to 10 piglets on day 1 of lactation, and maintained at this level until weaning on day 26 post-partum. Oestrus detection was performed daily, with oestrus defined as the exhibition of a standing reflex, and ovulation confirmed when progesterone concentrations exceeded 1.0 ng/ml on day 3 and 4.0 ng/ml on day 10 post-oestrus detection. There was no effect of PG600 on any reproductive measures, and no interactions with the BE treatment. A higher proportion of BE compared to non-BE sows exhibited a behavioural oestrus without ovulation within 5 days of parturition (0.83 versus 0.09; P < 0.05). The interval from parturition to lactation ovulation was unaffected by treatment (15.4 +/- 1.21 days). However, BE increased the proportion of sows ovulating during lactation (0.61 versus 0.09; P < 0.05), and resulted in an earlier ovulation relative to parturition (20.6 +/- 1.30 versus 30.1 +/- 1.35 days) compared to non-BE sows. In conclusion, this is the first study to demonstrate that taking lactating sows to a detection mating area for 15 min of daily, physical boar contact resulted in a high incidence of lactation ovulation within 22 days of parturition. (C) 2013 Published by Elsevier B.V.

This study evaluated the effect of full physical boar exposure and split weaning on the incidence of lactation estrus within a large commercial piggery. A total of 299 multiparous (MP; parity 2.5 ± 0.03) and 303 primiparous (PP) sows of Large White × Duroc × Landrace genetics were individually housed in conventional farrowing crates from 1 wk before expected farrowing until weaning on Day 30.7 ± 0.05 postparturition. Before shed entry, sows were allocated randomly within parity to receive either boar exposure (BE; n = 454) or no BE (No BE; n = 149). Sows assigned to receive BE were then allocated to 1 of 2 litter size treatments: litter size unchanged (BE; n = 302) or BE and the litter permanently reduced (split weaned) to 7 piglets (BESPW7; n = 152) on Day 18 of lactation. From Day 18 of lactation until weaning, sows in both BE treatments were taken daily to a detection mating area where they received 15 min of full physical BE and were artificially inseminated at the first observed estrus. Providing sows with BE increased the incidence of lactation estrus, with a further increase observed when litter size was reduced to 7 piglets (16% No BE vs. 62% BE and 75% BESPW7; P < 0.05). Multiparous sows exhibited a greater incidence of lactation estrus than PP sows irrespective of treatment (81 compared to 52%, respectively; P < 0.05). Both MP and PP sows exhibited an increased incidence of lactation estrus when a portion of the litter was removed (MP: 76 vs. 89% and PP: 47 vs. 61%; P < 0.05). Farrowing rates were higher in BE MP sows mated postweaning and all BESPW7 sows mated postweaning when compared to their counterparts mated in lactation (P < 0.05). Percentage live weight loss over the course of lactation was greatest for sows in the No BE compared to the BE and BESPW7 treatments (7.7% ± 0.5 vs. 5.4% ± 0.3 and 4.5% ± 0.4, respectively; P < 0.05). Between Day 17 and weaning, piglets suckling sows in the BESPW7 treatment had a higher average weight gain than piglets suckling sows with a full litter (3.5 ± 0.06 vs. 3.1 ± 0.05 kg; P < 0.05). In conclusion these data suggest that providing MP sows with BE is effective at stimulating a synchronous lactation estrus while PP sows require, in addition to BE, a reduction in suckled litter size to 7 piglets.

Within gilt pools, incidences of delayed puberty attainment, failure to exhibit regular oestrous cycles and low first litter size are often high. Boar exposure is an effective method of accelerating puberty; however, the timing of gilt response can vary greatly. Although, PG600 (400 IU of PMSG and 200 IU of hCG; Intervet) can induce a rapid and synchronous ovulatory response, thus providing an alternative to boar contact, the quality of the response is often variable. This study compared the effect of PG600, either alone (NBC) or in conjunction with boar exposure (BC), on puberty attainment and maintenance of oestrous cyclicity. The effects of first mating these gilts at the hormonally induced (pubertal) or second oestrus on ovulation rate and early embryo survival were also studied. Eighty Large White cross terminal (Duroc) line gilts were used in this study. The study was conducted in two blocks, with 10 gilts allocated to each of the four treatments in each block. Gilts were artificially inseminated at the allocated oestrus, with the reproductive tracts collected at 26.5+/-0.29 days after first mating (mean+/-S.E.M.), and the number of corpora lutea and viable embryos recorded. Mean days-to-puberty was significantly reduced (P<0.05) when gilts received both PG600 and boar exposure as opposed to PG600 alone (5.7+/-0.15 versus 6.9+/-0.37 days; P<0.01). The proportion of gilts exhibiting an ovulatory response to PG600 was similar for the BC and NBC treatment groups (0.88 and 0.84); however, the proportion of gilts exhibiting visible signs of oestrus in response to PG600 was significantly higher for the BC compared to the NBC treatment groups (0.81 versus 0.49; P<0.05). Boar contact resulted in a numerical, but not significant, increase in the proportion of gilts exhibited a second oestrus (1.00 versus 0.76). There was no significant effect of boar contact on ovulation rate, embryo number or survival. Although ovulation rate was unaffected by oestrus at mating, embryo number was significantly increased (P<0.05) following mating at the second compared to the first oestrus (11.2+/-0.96 versus 7.8+/-1.17). In conclusion, the current data indicate that the timing of puberty attainment and oestrus detection are significantly improved when PG600 treated gilts receive full boar contact. Further, it is evident that mating gilts at their second as opposed to the hormonally induced oestrus significantly increases embryo number at day 26 post-mating.

There is general acceptance that mixing sows during the first 3 weeks of gestation is detrimental to embryo development and survival. However, there is a paucity of data describing the influence of group housing and remixing during the first 14 days of gestation on pregnancy outcomes. Using 96 purebred maternal (Large White)/terminal (Duroc) line gilts, the current study determined the effects of regrouping, and the timing of regrouping, during the pre-implantation period on embryo mortality. The study was conducted in 2 blocks, with 12 gilts allocated to each of 4 treatments in each block. At 175 days of age, the combination of PG600 and 20 min of daily physical boar contact was used to stimulate puberty, with boar contact resuming 12 days after first detection of oestrus and gilts receiving two artificial inseminations (AIs), 24 h apart, at their second oestrus. After their first AI gilts were allocated to one of four treatment groups (n=12 gilts/treatment). Gilts in one treatment group were housed individually in stalls (STALL). The remaining gilts continued to be housed in their pre-AI groups and were either not remixed (NOMIX), or remixed to form new groups on day 3/4 (RMIXD3/4) or day 8/9 (RMIXD8/9) of gestation (day 0=day of first detection of second oestrus and first insemination). Group-housed gilts were housed in groups of 6, with a space allowance of 2.4 m2/gilt. All gilts were fed once a day (2.2 kg/gilt). Reproductive tracts were collected on day 26.6+/-0.13 of gestation, and the number of corpora lutea (CL) and viable embryos counted. Pregnancy rate was similar across all treatments, averaging 94.5% across the four treatment groups. The number of embryos present on day 26 of gestation was unaffected by housing treatments (P>0.05); gilts in the STALL, NOMIX, RMIXD3/4 and RMIXD8/9 groups possessed 13.2+/-0.67, 12.9+/-0.66, 14.1+/-0.46 and 13.8+/-0.57 embryos, respectively. Similarly, embryo survival rates were 0.91+/-0.04, 0.85+/-0.04, 0.91+/-0.02 and 0.87+/-0.05 for the STALL, NOMIX, RMIXD3.4 and RMIXD8/9 groups, respectively (P>0.05). In conclusion, the current data indicate that individually housing gilts immediately after their first AI does not improve embryo survival. There also appear to be no adverse effects on embryo development or survival when group-housed, mated gilts are remixed during the first 10 days of gestation.

Conducted during the Australian summer, this experiment evaluated the reproductive performance of sows receiving a diet supplemented with betaine, a potent organic osmolyte and methyl donor. Large White/Landrace/Duroc sows (n=450) ranging in parity from 1 to 7 (parity 2.9 ± 0.10, mean ± SEM), and mated between the 11th of January and 11th February were used. The treatments compared the effects of two gestation diets (standard (Stand) compared to betaine (Bet) supplemented) and two parity groups (parities one and two (P1/2) versus parity three and greater (P3+) on pregnancy outcomes and litter size. The betaine diet was fed from d 3 ± 1 post-mating until farrowing, with betaine content of the diet altered during gestation to ensure a daily intake of 7.6-9.0 g/sow. Liveweight (LW) and LW gain were unaffected by gestation diet; however, on d 1 of lactation P2 backfat (P2) tended (P=0.07) to be greater for standard compared to betaine fed sows (22.5 ± 0.42 compared to 21.5 ± 0.42 mm). P2, LW and LW gain were greater (P<0.05) for P3+ compared to P1/2 sows. Sow farrowing rate (0.79) was unaffected by gestation diet. Total litter size was greater (P<0.05) for Bet3+ (13.6 ± 0.35) sows compared to Stand3+ (12.1 ± 0.34), BetP1/2 (12.1 ± 0.36) and StandP1/2 (12.3 ± 0.38) sows. In conclusion, this is the first study to demonstrate that gestational betaine supplementation during summer increased litter size of sows with greater numbers of parities.

ABSTRACT Maternal intake of B-vitamin and methyl donors can affect sow prolificacy. A total of 1079 Large White/Landrace sows (parities 2-9 at mating) were used in a 2 by 2 by 2 factorial design to determine the effects of two levels of betaine supplementation (0 versus 3 g added betaine/kg feed), two levels of folic acid plus vitamin B-12 supplementation (0 versus 20 mg/kg folic acid plus 150 mu g/kg vitamin B-12) during gestation, and two parity groups (parity 2 and 3 versus parity 4 and greater) on litter size and pregnancy outcomes. The number of sows returning to oestrus post-insemination, as well as the number of early (<Day 30) and late (>Day 30) pregnancy losses were recorded. At farrowing, the total number of piglets born, the number of piglets born alive and dead, as well as the number of mummified fetuses were recorded. Pre-prandial blood samples were collected from a subset of 20 sows/treatment on Days 3, 30 and 107 of gestation to analyse homocysteine. The incidence of early pregnancy loss was reduced (P < 0.001) by folic acid plus vitamin B-12 supplementation (0.03 versus 0.07). There was a significant interaction between parity at mating (parities 2 and 3 versus parity 4 and greater) and the addition of betaine or folic acid plus vitamin B-12 to the gestation diet on litter size. Litter size was higher (0.5 piglets; P < 0.05) for betaine supplemented, compared with unsupplemented, parity 4 plus sows. Folic acid plus vitamin B-12-supplemented parity 2 and 3 sows gave birth to more (P < 0.05) piglets than all other treatment groups. Folic acid plus vitamin B-12 supplementation decreased (P < 0.001) plasma homocysteine concentration by 2.2 and 2.8 mu M, respectively, on Days 3 and 107 of gestation. However, betaine supplementation decreased (P < 0.05) homocysteine on Day 3 only. Overall, folic acid plus vitamin B-12 supplementation decreased incidences of early pregnancy failure and increased litter size in early parity sows, while betaine increased litter size in older parity sows. Received 14 December 2011, accepted 4 March 2012, published online 2 October 2012

Inclusion of high levels of the high-fibre ingredient sugar-beet pulp in pre-mating diets has been shown to increase gonadotrophin concentrations and improve oocyte quality in nulliparous pigs (gilts). This study evaluated the effects of two alternative fibre sources on reproductive performance in gilts. Gilts received one of three diets from 3 weeks before puberty stimulation until Day 19 of the first oestrous cycle: control (39 g kg⁻¹ fibre), bran (500 g kg⁻¹ wheat bran, 65 g kg⁻¹ fibre) or lupin (350 g kg⁻¹ lupin, 118 g kg⁻¹ crude fibre). Diet did not affect circulating LH concentrations or ovarian follicle size. However, a higher percentage of oocytes collected from lupin-supplemented gilts reached metaphase II in vitro compared with those collected from bran-fed or control gilts (89±5% versus 72±5% and 66±5%, respectively; P<0.05). Furthermore, in a second experiment, gilts fed the same lupin-based diet before mating had improved embryo survival (92±5%) on Day 28 after mating compared with control gilts (76±4%; P<0.05). Therefore, feeding a high-fibre diet before mating can improve oocyte quality in gilts without changes in circulating LH, but this effect is dependent on the fibre source.