A Short-Term Altrenogest Treatment Post-weaning Followed by Superovulation Reduces Pregnancy Rates and Embryo Production Efficiency in Multiparous Sows

Abstract

Although embryo transfer (ET) is a biotechnology ready for the swine industry, there are factors to be solved, the availability of embryo donors as one. Multiparous sows as donors ought to be considered since weaning is a natural and efficient method for estrus synchronization. In addition, superovulation treatments at weaning are effective in increasing the efficiency of donor embryo production. However, ET programs typically require more donors than those available from a single weaning, imposing grouping several weanings to establish a batch for ET. Since short-term administration of Altrenogest is effective in delaying estrus after weaning without effects on ovulation and embryo development, we investigated how Altrenogest combined with superovulation would affect reproductive parameters and embryo quality and quantity of weaned multiparous donor sows. The sows were administered Altrenogest from the day of weaning for 14 (SS-14 group; N = 26), 7 (SS-7 group; N = 31) and 4 (SS-4 group; N = 32) days. The sows were superovulated with eCG 24 h after the last administration of Altrenogest and with hCG at the onset of estrus. Sows not treated with Altrenogest that were superovulated with eCG 24 h post-weaning and hCG at the onset of estrus (SC group; N = 37) and sows with natural estrus after weaning (C group; N = 34) were used as control groups. The percentage of sows showing estrus within 10 days was not affected by the treatment, but the interval from Altrenogest withdrawal to estrus was longer (P < 0.05) in the SS groups than the interval from weaning to estrus in the controls. SS treatments increased (P < 0.05) the percentage of sows with ovarian cysts and the development of polycystic ovaries. The pregnancy and the fertilization rates, and the overall embryo production efficiency were also negatively affected by the SS treatments (P < 0.05). Interestingly, almost 70% of the structures classified as unfertilized oocytes or degenerated embryos in sows from the SS groups were immature oocytes. In conclusion, although superovulation of weaned sows was highly efficient, short-term administration of Altrenogest in combination with superovulation had negative effects on most of the reproductive parameters assessed, particularly affecting the overall efficiency of pregnancy and embryo production.

Keywords: Altrenogest; embryo; embryo transfer; estrus synchronization; pig; superovulation; weaning.

Figure 1

Occurrence of estrus and pregnancy rates in weaned sows superovulated after Altrenogest treatment for 14 (SS-14 group; N = 26), 7 (SS-7 group; N = 31) and 4 (SS-4 group; N = 32) days. Superovulated weaned sows without prior Altrenogest treatment and weaned sows with natural estrus were used as controls (SC group, N = 37 and C group, N = 34, respectively). (A) Percentage of sows in estrus within 10 days of the end of treatment or weaning. (B) Box plots showing the interval from the last Altrenogest feeding (SS-14, SS-7, and SS-4 groups) or weaning (SC and C groups) to the onset of estrus. Values are given as medians (thick lines) and interquartile ranges (boxes, Q1–Q3); triangles and circles represent the mean and outliers, respectively. ^a,b Different letters indicate significant differences (p < 0.01). (C) Pregnancy rates 6 days after the onset of estrus in sows from the different groups. Sows with at least four viable embryos were considered potentially pregnant. The pregnancy rate was calculated as the ratio of the number of pregnant sows to the number of sows in estrus or to the total number of sows used. ^a,b Different letters within each variable indicate differences (p < 0.05).

Figure 2

Reproductive disorders in weaned sows superovulated after Altrenogest treatment for 14 (SS-14 group; N = 26), 7 (SS-7 group; N = 31) and 4 (SS-4 group; N = 32) days. Superovulated weaned sows without prior Altrenogest treatment and weaned sows with natural estrus were used as controls (SC; N = 37 group and C; N = 34, respectively). (A) Percentage of sows with cysts and the number of cysts per sow in sows that showed signs of estrus after SS treatments or weaning. ^a,b Different letters within each variable indicate differences (p < 0.05). (B) Representative images of a polycystic ovary at day 6 after the onset of estrus with at least four large follicular cysts (asterisks) and the absence of corpora lutea. (C) Representative images of ovaries with numerous corpora lutea (arrows) and several small follicular cysts (asterisks).

Figure 3

(A) Findings of oocyte immaturity in structures classified as unfertilized oocytes and degenerated embryos recovered from weaned sows superovulated after treatment with Altrenogest. The number of structures evaluated was 25, 23, 21, 19, and 16 for groups SS-14, SS-7, SS-4, SC, and C, respectively. ^a,b Different letters within each variable indicate significant differences (p < 0.01). (B) Unfertilized immature oocytes at the germinal vesicle stage (arrow). (C) Unfertilized mature oocyte with chromosomes at metaphase stage II (arrow) and the first polar body (arrowhead). (D) Embryos classified as degenerate showed irregular morphology and a low cell number after staining with Hoechst 33342.

Figure 4

Differential staining of embryos at the blastocyst stage. (A) Hoechst-stained nuclei of all blastomeres (blue) and anti-CDX2-stained trophectoderm (TE) cells (red). Merged images show inner cell mass (ICM) and TE cells with blue and pink-red fluorescence, respectively. (B) Total cell number (TCN) and ICM/TCN ratio in blastocysts collected at day 6 after the onset of estrus from weaned sows superovulated after Altrenogest treatment for 14 (SS-14 group; N = 10), 7 (SS-7 group; N = 12) and 4 (SS-4 group; N = 12) days. Superovulated weaned sows without previous Altrenogest treatment and weaned sows with natural estrus were used as controls (SC, N = 14; and C, N = 12).

Figure 5

Detection of apoptosis in embryos at the morula stage collected on day 6 after the onset of estrus in weaned sows superovulated after Altrenogest treatment for 14 (SS-14 group; N = 15), 7 (SS-7 group; N = 16) and 4 (SS-4 group; N = 16) days. Superovulated weaned sows without prior Altrenogest treatment and weaned sows with natural estrus were used as controls (SC, N = 17; and C, N = 15 groups, respectively). (A) Representative fluorescence images of the TUNEL assay (including TUNEL-positive and TUNEL-negative controls). The blue Hoechst signal (left), green TUNEL staining (middle), and a merged image (right) are shown. (B) Percentage of morulae with apoptotic cells. (C) Number of blastomeres and apoptosis rates in embryos from the different groups.

Figure 6

Cryosurvival and embryo development after vitrification and warming. (A) In vitro survival and embryonic developmental stage after 24 h culture of vitrified and warmed morulae. Fresh morulae were collected from weaned sows superovulated after Altrenogest treatment for 14 (SS-14 group; N = 27), 7 (SS-7 group; N = 60) and 4 (SS-4 group; N = 65) days. Superovulated weaned sows without previous Altrenogest treatment and weaned sows with natural estrus were used as controls (SC, N = 68; and C; N = 58). Developmental stage was scored according to the following classes: 1, morulae; 2, early blastocysts; 3, blastocysts; and 4, hatching or hatched blastocysts. (B,C) Representative images of morulae before vitrification and 24 h after warming, respectively.