Bacillus subtilis QST 713 Supplementation during Late Gestation in Gilts Reduces Stillbirth and Increases Piglet Birth Weight

Recent studies have shown that probiotic supplementation during late gestation exerts some beneficial effects on reproductive performance of the sows. This study aimed to investigate effects of Bacillus subtilis QST 713 supplementation in gilts on different reproductive criteria. A total of 94 Camborough-48 gilts at day 85 of gestation were randomly allocated into 2 groups: (1) control diet; (2) control diet + 4 × 108 CFU Bacillus subtilis QST 713 per day. Gilts were supplemented until farrowing. At farrowing, litter size, number of piglets born alive, stillbirths, mummies, birth weight, farrowing duration, and birth interval were recorded. Within litter variation of piglet birth weight, depicted as SDBW and CVBW, was also calculated. Results showed that Bacillus subtilis QST 713 supplementation decreased stillbirth rate (1.26 vs. 4.37%, p=0.035) and increased birth weight of the piglets (1303.94 vs. 1234.09 g, p=0.007). Also, the litter size (11.85 vs. 10.67, p=0.03), number of piglets born alive (11.71 vs. 10.23, p=0.008), and litter weight (15473.06 vs. 13174.86 g, p=0.002) in the treatment group were higher than those in the control. Farrowing duration (174.39 vs. 160.81 minutes, p=0.162), birth interval (16.32 vs. 16.59 minutes, p=0.674), SDBW (85.07 vs. 94.65 g, p=0.343), and CVBW (6.42 vs. 7.85, p=0.12) were independent of the Bacillus subtilis QST 713 supplementation. Results of the present study indicate that supplementation of Bacillus subtilis QST 713 during late gestation in gilts reduces stillbirth and increases birth weight thereby improving their reproductive performance.


Introduction
During late gestation, the richness of microbiota in intestine of the sows reduced [1,2] while fecal endotoxin increased [1]. Besides, an increase in gut permeability results in increased plasma endotoxin concentration in sows [1]. ese changes may influence sows' digestion, absorption, and nutrient metabolism which may affect the birth weight of piglets [3].
Bacillus subtilis QST 713 used in this study was isolated from soil in California, USA, in 1995. is strain is reported to be low toxic to mammals and not likely to be a pathogenic agent in human. Supplementation of Bacillus subtilis QST 713 was suggested to be an effective approach to control necrotic enteritis in broilers [11]. Furthermore, Bacillus subtilis QST 713 increased the length of intestinal villi and decreased the number of deep crypts [12]. Bacillus subtilis QST 713 supplementation also suppressed the harmful effect of antimicrobial on beneficial bacteria in the intestine of the weaned pigs thereby working as "a preventative and/or attenuating compound against dysbiosis in piglets" [13]. All of those findings suggest that Bacillus subtilis QST 713 may increase feed digestion and nutrients absorption in animals. Based on available reports in beneficial effects of Bacillus subtilis QST 713 in animals, we hypothesized that this strain might improve reproductive performance of the pig. e reason to this hypothesis was that the increased nutrients absorption might lead to the increased birth weight and subsequently decreased stillbirth rate in newborn piglets. To this end, the present study aimed to evaluate effects of Bacillus subtilis QST 713 supplementation from day 85 of gestation to farrowing on different reproductive criteria including the number of piglets born alive, litter birth weight, stillbirth rate, dead born rate, birth weight, within litter variation of piglet birth weight, farrowing duration, and birth interval.

Animal Care.
e present experiment was reviewed and approved by the Committee on Animal Research and Ethics of Faculty of Veterinary Medicine, Vietnam National University of Agriculture (CARE-2021/01).

Animals, Diet, and Experimental Design.
is study was conducted in a commercial pig farm during February and July, 2021 in Bavi, Hanoi, Vietnam, enrolling ninety-four Camborough-48 gilts. Young gilts were vaccinated against porcine reproductive and respiratory syndrome, Aujeszky's disease, classical swine fever, foot and mouth disease, and porcine circovirus during 24-29 weeks of age. At the age of 30 weeks, estrus gilts were bred with diluted semen of PIC ® 337 boars. Pregnant gilts were vaccinated against classical swine fever, foot and mouth disease, and Escherichia coli. During the first 84 days of gestation, gilts were fed 1.8-2 kg of a gestation feed (GF07, Greendfeed Vienam). From days 85 to 110, gilts were fed 2.0-2.2 kg of a lactation feed (GF08, Greenfeed, Vietnam). Between day 111 and farrowing gilts received 1.0-1.5 kg of the lactation feed. e nutrient composition of the gestation and lactation feed was shown in Table 1. At day 85 of gestation, 94 gilts were randomly assigned into 2 groups with 47 each. In the treatment group, gilts were daily supplemented with 4 × 10 8 CFU Bacillus subtilis QST 713 (Baymix, GROBIG ® BS, Bayer de Mexico, S.A. de C.V., Mexico) in 4 g mixture of the probiotic and dextrose, which was topped to the daily feed (GF08, Greedfeed, Vietnam). Gilts in the control group received 4 g dextrose. Pregnant gilts were individually housed in gestation crates, and moved to farrowing crates at about day 107 of gestation. All animals were ad libitum accessed to water provided through a bite nipple drinking system.

Data Collection.
At parturition, gilts were supervised by two trained veterinarians. Litter size (total born), number of born alive piglets, dead born piglets (stillbirth and mummy), gestation length, birth interval, farrowing duration, and birth weight were recorded. Mummified piglets were those born dead with full brown/black color of skin. Other born dead piglets were classified as stillbirth. Stillborn piglets included intrapartum and prepartum stillbirths. However, postmortem examination was not allowed due to the regulation of the farm then these two types of stillbirth were not distinguished. Birth interval was the time period between the births of two consecutive piglets. Farrowing duration was the interval between the birth of the first piglet and the birth of the last piglet. Piglets were individually weighed with a 5 g precision digital scale before colostrum intake. Mean birth weight (MBW) of each litter was calculated by dividing litter weight by litter size. Standard deviation of mean birth weight of each liter (SDBW) was also calculated. Coefficient of variation of birth weight of each litter (CVBW) was derived by dividing SDBM by MBW then multiplying with 100.

Statistical Analysis.
To deal with hierarchical data where multiple piglets born from a sow, generalized linear mixed model was used to compare the stillbirth rate and dead born rate of piglets in the treatment and control groups. Similarly, linear mixed effect model was used to compare piglet birth weight and birth interval between treatment and control groups. Litter size, number of piglets born alive, litter weight, SDBW, CVBW, and farrowing duration were compared between treatment and control groups using a t-test. All tests were conducted in RStudio Desktop 1.3.1093 (Boston, MA, RStudio Team: Integrated Development for R). A p value <0.05 was considered significant.

Discussion
e present study revealed that Bacillus subtilis QST 713 supplementation both decreased the stillbirth rate and increased the birth weight. Interestingly, the increased birth weight was simultaneously present with a higher litter size and higher number of piglets born alive. Moreover, despite the larger litter size, the within litter variation of piglet birth weight depicted as SDBW and CVBW in the treatment group was numerically lower than that in the control group. e larger litter size in the treatment group in this study reflected the results of some previous studies [5,8]. Other studies also reported a numerically higher litter size in probiotic supplementation groups in comparison with that in control groups [6,7]. However, as discussed by Zhang et al. [5] the increased litter size in the treatment group is independent of probiotic supplementation because the litter size had already been determined before the use of probiotics. By contrast, the number of piglets born alive (NBA) which is the result of total born minus the number of piglets born dead (NBD) might be influenced by the probiotic supplementation. e increased NBA in the treatment group was in agreement with some previous findings [5,8,9]. However, many other studies reported nonsignificant effect of probiotic supplementation on this criterion [4,6,7,10,[14][15][16][17][18][19][20]. e positive effect of the probiotic supplementation on NBA may be partly attributable to the fact that it reduced stillbirth rate. Previous studies demonstrated that probiotic supplementation did not reduce piglet stillbirth rate [4][5][6][7][8][9]. It has been shown that stillbirth decreased when birth weight increased [21][22][23][24]. erefore, the reduced stillbirth rate in the treatment group may be due to the increased birth weight (1303.9 ± 161.5 g vs. 1234.1 ± 137.3 g).
It is interesting that Bacillus subtilis QST 713 increased both litter size/NBA and birth weight despite the fact that birth weight is negatively associated with litter size [25,26]. Some previous studies failed to find any beneficial effects of probiotic supplementation at late gestation on birth weight of piglets [7][8][9]20] while other studies even found a reduced birth weight in the probiotic supplementation group where litter and/or NBA increased in comparison with that in the control [5,14]. Two previous studies found that birth weight was increased while litter sizes were unaltered when probiotic was supplemented throughout 2 successive estrus cycles [27] or late gestation [28]. However, in the former study, the positive effect of probiotic on birth weight only exhibited in the second estrus cycle [27], and in the latter one, the increased birth weight seemed to be attributable to isomaltooligosaccharide rather than probiotic [28].
It is well documented that the litter size positively correlates with within litter variation of piglet birth weight [29][30][31]. In other words, a larger litter size results in a larger variation of piglet birth weight. However, in the present study, the probiotic supplementation increased litter size while did not increase within litter variation of piglet birth weight. is finding can be partly explained via the increased birth weight in the treatment group because birth weight has been found negatively associated with CVBW [29].
In the present study, the effect of Bacillus subtilis QST 713 supplementation on farrowing duration and birth interval is diverse from the finding of the solely existed study that evaluated this aspect [5]. In their study Zhang et al. [5] speculated that the sows and piglets in the treatment group might be more physically stronger, therefore, the farrowing duration and birth interval were reduced. It is well established that litter sizes are positively associated with farrowing duration [26,32], and birth weight positively correlated with birth interval [25]. erefore, the increased litter size and birth weight might mask any potentially beneficial effect of Bacillus subtilis QST 713 supplementation on farrowing duration and birth interval resulting in nonsignificant difference in the present study. e mechanism of action of Bacillus subtilis QST 713 supplementation on gilt reproductive performance is not totally clear. Previous studies showed that Bacillus subtilis improve the intestinal immune status [33], and immunological response to vaccination [34,35]. Bacillus spp. can produce a wide range of antimicrobial substances which were found to inbibit the growth of many pathogenic bacteria such as Clostridium difficile, Campylobacter jejuni, Streptococcus pneumoniae, Campylobacter coli, Proteus vulgaris, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Salmonella typhi [36,37]. ese inhibitions might lead to lowered sera endotoxin concentration in sows supplemented with Bacillus subtilis during the late stage of gestation [5]. Tactacan et al. [11] reported that Bacillus subtilis QST 713 could also produce some antimicrobials although the names of these products were not specified. In chicken, Bacillus subtilis QST 713 stimulated the development of intestinal mucosa and villi, and the growth of Lactobacillus spp., and inhibited Escherichia coli and Enterococcus spp. [12]. In the pig, Bacillus subtilis QST 713 prevented the Veterinary Medicine International growth of Escherichia coli, and promoted the development of beneficial bacteria such as Bulleidia [13]. e beneficial effects of Bacillus spp., Bacillus subtilis, and Bacillus subtilis QST 713 lead to the suggestion that supplementation of Bacillus subtilis QST 713 during the late gestation might enhance the intestinal immune status and the health of gut microbiota, increase the digestion and absorption of nutrients in treated gilts. ese promoting effects resulted in enhanced nutrient delivery to the swine fetuses which subsequently increased birth weight, and decreased stillbirth rate.
e present study had a limitation. We used commercially industrialized diets in which only some main ingredients are listed without any further information. is led to the difficulty of formulation of such diets in the future studies. Nevertheless, the promising beneficial effect of Bacillus subtilis QST 713 on reproductive performance of gilts/sows deserves further investigation in the future.

Conclusion and Recommendations
e present study suggested that supplementation of Bacillus subtilis QST 713 at the dose of 4 × 10 8 cfu/meal/day from day 85 of gestation to farrowing in gilts could decrease stillbirth rate and increase the birth weight. e increase birth weight and decreased stillbirth rate were simultaneously present with a larger litter size/NBA. Bacillus subtilis QST 713 is a promising probiotic supplement in gilts during late gestation for improvement of reproductive performance.
Data Availability e data can be acquired from the principal author upon reasonable request.

Conflicts of Interest
e authors declare no conflicts of interest. e results are presented as mean ± standard deviation; p: probability. Litter size included live born, stillborn, and mummified piglets. Dead born piglets included stillborn and mummified piglets. SDBW: standard deviation of mean birth weight of litter. CVBW: coefficient of variation of mean birth weight of litter. 4 Veterinary Medicine International