In Nulliparous and Multiparous Ovariectomized Goats Is Possible to Induce Maternal Behavior with Hormonal Treatment Plus Vagino-Cervical Stimulation

Simple Summary

We evaluated whether ovariectomized nulliparous or multiparous goats treated with estradiol benzoate or progesterone + estradiol benzoate, before or after vagino-cervical stimulation, exhibited maternal behavior. Goats were tested for 30 min in the presence of an alien kid, which was one day old. Both nulliparous and multiparous goats treated with estradiol after vaginal stimulation emitted more maternal bleats and spent longer cleaning the offspring, while locomotor activity was more frequent before vaginal stimulation. Nulliparous goats with estradiol emitted more maternal bleats after vaginal stimulation than before, while locomotor activity was more frequent before vaginal stimulation than after. Multiparous goats with estradiol exhibited a higher frequency of aggression and sniffing of the kids before vaginal stimulation than those treated with progesterone + estradiol. Within treatments, the estradiol group emitted more low-pitch bleats and cleaned the kids more after vaginal stimulation than before; the progesterone + estradiol group smelled the kids more after vaginal stimulation than before. The maternal index after vaginal stimulation was higher in goats with estradiol. Nulliparous goats treated with progesterone + estradiol had a higher maternal index compared with those treated with estradiol alone. In conclusion, treatment with estradiol, combined with vaginal stimulation, is more effective in inducing maternal behavior, particularly in multiparous goats.

Abstract

No studies have induced maternal behavior in goats through hormonal treatment. We evaluated whether ovariectomized goats treated with estradiol benzoate (EB2; n = 7 nulliparous and 10 multiparous goats) or progesterone + estradiol benzoate (P4 + EB2; n = 7 nulliparous and 7 multiparous goats), before or after vagino-cervical stimulation (VCS), displayed maternal behavior. When all goats were measured within treatments, in the EB2 group, low-pitch bleats were more frequent, and the time spent cleaning the kids was longer after VCS (p < 0.01), while location changes were more frequent before VCS. In nulliparous goats, those in the EB2 group emitted more low-pitch bleats after VCS than before (p = 0.04). The frequency of location changes was higher before VCS than after (p = 0.05). In multiparous goats, the frequency of smelling the kids and aggression toward the kids before VCS was higher in the EB2 group than in the P4 + EB2 group (p < 0.01). Within treatments, the EB2 group emitted more low-pitch bleats after VCS than before (p = 0.01), and the duration of cleaning the kids was longer after VCS than before (p = 0.028). Within the P4 + EB2 group, the kids were smelled more frequently after VCS than before (p = 0.03). The maternal index after vagino-cervical stimulation was higher in goats with EB2 (p = 0.002). Nulliparous goats treated with P4 + EB2 had a higher maternal index compared with those treated with EB2 alone (p = 0.04). In conclusion, regardless of parity, maternal behavior induced with the EB2 treatment was better when VCS was applied. Likewise, maternal experience altered the response. In multiparous females, any of the treatments were better after VCS, while in nulliparous females, this was only evident with the EB2 treatment.

1. Introduction

It has been demonstrated that mother–kid bonding in goats is established during the first 4 h postpartum [1]. The females show maternal behavior immediately after fetal expulsion, as they are highly attracted to the amniotic fluid surrounding the neonate; this attraction causes intense licking and sniffing of their offspring [2]. Maternal behavior in certain mammalian species, such as rabbits, sheep, and rats is modulated by hormones, such as the increase in the plasma concentration of estrogens produced days before delivery, the fall in concentration of progesterone, the participation of oxytocin at the central nervous system level, and prolactin in the peripheral nervous system, all of which triggering maternal behaviors of care and protection [3,4,5,6,7]. Few studies have investigated the role of hormones in maternal behavior in goats; these studies have only evaluated the relationship between the concentrations of some steroid hormones and the presence of certain maternal behaviors [8]. To our knowledge, no studies have investigated whether it is possible to induce maternal behavior in goats through hormonal treatment. Most research on this topic has assumed that the hormonal control of maternal behavior in goats is similar to or equal to that in sheep, although there are no studies to support this assumption. Furthermore, the fact that maternal behavior in goats is regulated by the principle of hiding behavior has been omitted [9], since in domestic ungulates, it is known that the paradigm hiding/following is the one that could regulate the mother–offspring relationship [10], where sheep are considered followers and goats are considered hidden. Therefore, it is necessary to research goats in this regard.

In ewes, high concentrations of pre-lambing estrogens help trigger maternal behavior [11]; likewise, the progesterone concentration drops three days before lambing, and remains so in the postpartum period, while estrogen concentrations rise three days before lambing, reaching their highest concentration during lambing [7,12,13].

In addition to the roles played by progesterone and estrogens, oxytocin is released during parturition via cervicovaginal stimulation [14,15,16,17,18,19,20]. Estrogens promote the formation of specific receptors for oxytocin before lambing [3,7,21]. However, in ewes, maternal behavior may be influenced by maternal experience, with primiparous ewes showing more rejection behaviors toward their offspring than multiparous ewes [22].

Cervical–vaginal stimulation (CVS) caused by fetal expulsion plays an important role in establishing maternal selectivity in both ewes and goats [23,24]. In goats, epidural anesthesia applied at the first signs of parturition (early epidural, on average 1 h and 30 min before parturition) significantly affected maternal behavior, compared with when it was applied 15 min before fetal expulsion (late epidural) [24,25]. In the same study, the effects of blocking CVS did not differ between nulliparous and multiparous goats [24,25], which contrasts with the findings demonstrated in sheep. In addition, in goats, CVS favors the establishment of maternal bonding, enabling them to accept even strange kids [26].

In work carried out with ovariectomized multiparous and nulliparous ewes, in which maternal behavior was induced with exogenous estrogen hormones or estrogen plus progesterone, as well as vagino–cervical stimulation (VCS), both treatments induced maternal behavior, especially after VCS, and multiparous ewes showed a greater maternal behavioral response than nulliparous ewes [11]. Another study found that most of the ewes treated with progesterone alone were not maternal, and their maternal index was low regardless of VCS [27]. Conversely, VCS significantly increased the maternal score in ewes with short progesterone plus estrogen treatment [27]. However, to our knowledge, no studies have investigated protocols using exogenous hormones in combination with VCS to induce maternal behavior in goats. Therefore, we hypothesized that the application of estradiol or estradiol plus progesterone treatments in ovariectomized goats can induce maternal behavior, and the effects of these treatments can be enhanced with VCS. Furthermore, females with maternal experience may respond better to these induction protocols. Therefore, the objective of our study was to evaluate treatments with estradiol or estradiol plus progesterone, as well as the role of VCS in maternal behavior, in both nulliparous and multiparous goats.

2. Materials and Methods

2.1. Animals and Feeding and Maintenance Conditions

This study was conducted at the goat production unit of the Centro de Enseñanza Agropecuaria, Facultad de Estudios Superiores Cuautitlán, Universidad Nacional Autónoma de México (UNAM), located in the State of Mexico, Mexico.

Seven nulliparous goats (between 1 and 2 years of age, without any previous births) and ten multiparous goats (between 2 and 5 years of age, with at least one and up to four previous births) of French Alpine and Toggenburg crosses were used. These animals were tested in two consecutive years, alternating hormone treatments. Additionally, 12 multiparous goats and 8 nulliparous goats were used to obtain the offspring for maternal motivation tests. All goats were fed a diet containing commercial balanced feed (18% Crude Protein, Technical Nutrition^®), corn silage, oats, and hay alfalfa. This diet provided their nutritional requirements [28]. They were also provided water ad libitum.

The goats were housed in three pens (12 × 15 m) with concrete floors, an automatic water trough, and a canoe-type feeder (12 × 0.3 m), with a shaded area (12 × 6 m). The females that would provide the kids for the tests were subjected to a synchronization protocol similar to that described in previous studies in goats, to concentrate the births in a single week and facilitate the execution of the tests [8,29]. At approximately day 60 post-service, gestation diagnosis was performed using real-time ultrasound (WELLD, Shenzhen Well. D Medical Electronics, Welld 9618, 3.5 MHz, Shenzhen, China).

2.2. Experimental Process

2.2.1. Ovariectomies

All 17 goats underwent ovariotomies three months before the donor and intact goats gave birth. All goats were assessed for ASA (American Society of Anesthesiologists) 1–2 anesthetic risk and underwent ovariectomies under general anesthesia with 36 h of solid fasting and 24 h of liquid fasting. The procedure for performing the ovariectomies was similar to that described by Carroll et al. [30]. Likewise, the goats’ anesthetic management was monitored by a specialist at all times and was carried out in accordance with previous procedures for goats [31]. Once the surgeries were completed, the goats were placed in pens for recovery.

2.2.2. Experimental Groups

Hormone treatments were applied to each group progressively as the control females gave birth. The females were divided into two groups depending on the hormonal treatment:

(1)

In the estradiol only group (EB2), ovariectomized goats (7 nulliparous and 10 multiparous goats) were given 2 intramuscular injections of estradiol benzoate with 24 h intervals between each application (100 μ per injection/day, Lab. Syntex, Mexico City, Mexico) over 48 h.

(2)

In the progesterone + estradiol (P4 + EB2) group, ovariectomized goats (7 nulliparous and 7 multiparous goats) were treated with intravaginal sponges (for 11 days) with a progestogen known as cronolone (20 mg; Chronogest CR^®, Intervet Productions S. A., Igoville, France). A single dose of 100 μg of EB2 was injected intramuscularly, 12 h after sponge removal.

2.2.3. Parturition and Birth of Offspring for Maternal Motivation Tests

To ensure the offspring’s survival, the births of the mothers that would lend their offspring for the tests were supervised for two hours. It was verified that the female had fed the kid and that a filial bond had been established.

2.2.4. Maternal Motivation Tests on Ovariectomized Goats with Hormone Treatment

Tests were conducted 24 h after the last application of estradiol benzoate for either treatment (EB2 or P4 + EB2). All females were fed before the test. The maternal motivation test was divided into two phases, as described in Figure 1.

Maternal Motivation Test Before Vagino-Cervical Stimulation (VCS)

Once any of the two hormone treatments were completed, the female was placed in an individual 2 × 2 pen, which was set up inside the communal pen. During this test, the goat was always kept in the company of the herd to minimize stress caused by social separation.

The first part of the test lasted for 30 min, and the goat was exposed to the contact of an alien kid, which was taken from an adjoining pen where the goats that had given birth were located. The kids’ ages ranged from 6 h to 2 days [11,27]. The records were collected through direct observation using a focal method and were conducted by two individuals who remained consistent throughout. The behaviors contained in

Table 1. Behaviors recorded in the mothers during the maternal motivation test in the presence of an alien kid.

Behavior	Description	Unit of Measurement
Low-pitch bleats	Number of vocalizations emitted by the female with her mouth closed	Frequency
High-pitch bleats	Number of vocalizations emitted by the female with her mouth open	Frequency
Location changes	Number of times the goat changed position or crossed the quadrants painted on the floor	Frequency
Aggression toward kid	Number of times the female threatened, bit, or hit the kid	Frequency
Suckling acceptances	Number of times the goat allowed the kid to approach the udders for more than 5 continuous seconds and/or suckle	Frequency
Suckling refusals	Number of times the goat did not allow the kid to approach the udders and/or suckle	Frequency
Suckling duration	When the female allowed the kid to ingest milk, and it was only considered when the episode lasted for more than 5 continuous seconds	Duration (s)
Cleaning of kid	When the female cleaned the kid in a grooming manner	Duration (s)
Smelling of kid	When the goat brought its nose close to the kid to sniff it	Frequency

were recorded on a pre-formatted sheet for the mothers during the test. Once the first part of the test was completed, the female was released from the pen to rest and/or eat for 30 min, and the kid was taken to its mother in the herd pen.

Maternal Motivation Test After Vagino-Cervical Stimulation

Once the 30 min rest period was over, VCS was performed for 5 min outside the test pen using a technique previously described in goats [26]. Briefly, with a clean gloved hand, a natural lubricant was applied, and two fingers were inserted into the female’s vagina, gently making circles until the other fingers were gradually inserted, followed by the hand. Once inside, the fist was closed, and the hand was pushed and pulled continuously for 5 min.

Immediately after this procedure, the female was placed in the test pen and put into contact with a different kid from the one used in the first stage of the test. The observation lasted for 30 min, using the same process described above. Once the second part of the test was completed, the tested female was released into the pen with her mates, and the kid was reunited with its mother in the herd pen.

2.2.5. Maternal Motivation Index

To evaluate the response of hormonal treatments in treated goats, maternal criteria were considered, which led to the construction of a maternal motivation index [27,32] as follows: Briefly, the kid’s acceptance and rejection behaviors were standardized to have equal weight. The maternal index was calculated according to the formula indicated by Poindron et al. [31], by summing all the standardized acceptance behaviors and subtracting all the standardized rejection behaviors. The result was interpreted as follows: the higher the algebraic value, the more marked the kid’s acceptance, and a negative value for this index indicated rejection.

The acceptance behaviors were as follows: the number of sniffs of the kid, the duration spent cleaning the kid, the number of low-pitch bleats, and the number of suckling acceptances, and the duration of suckling. The rejection behaviors were as follows: the number of high-pitch bleats, the number of changes in place, the number of aggressions to the kid, and the number of refusals to suckle.

2.3. Statistical Analysis

This study was based on the individual goat as the experimental unit. The normality of the data was verified using a Kolmogorov–Smirnov one-sample test with the Lilliefors probability correction. The Mann–Whitney U test was used to compare the frequencies and durations of behaviors, as well as the maternal index among goats of each hormonal treatment. The comparison of frequencies and durations of behaviors, as well as the maternal index within each treatment, was analyzed using a Wilcoxon signed-rank test throughout the test phases (before and after VCS). A significance level of p ≤ 0.05 was established. Statistical analyses were performed with the SYSTAT 13 program (SPSS Inc., Chicago, IL, USA). The results are expressed as mean ± SEM.

3. Results

3.1. Tests of Maternal Motivation Compared Between Treatments Without Considering Parity

When comparing treatments without considering parity and during testing after VCS, it was found that the duration of cleaning the kid was longer in goats in the EB2 treatment (47.5 ± 39.9 s) than in the P4 + EB2 group (1.5 ± 1.2 s) (p = 0.050).

3.2. Tests of Maternal Motivation Compared Within Treatments Without Considering Parity

In the EB2 group, low-pitch bleats were more frequent after VCS than before VCS (p = 0.001; Figure 2), while high-pitch bleats tended to be more frequent before than after VCS (p = 0.10). The time spent cleaning the kids was longer after VCS than before VCS (47.5 ± 39.9 vs. 0.11 ± 0.11 s; p = 0.018). Location changes were more frequent before VCS than after VCS (p = 0.028). Finally, suckling acceptances tended to be more frequent after VCS than before VCS (p = 0.059). In the P4 + B2 group, no differences were observed in any behaviors recorded during this treatment when goats were compared before and after VCS (p > 0.05).

3.3. Comparison Between Treatments in the Maternal Motivation Test in Nulliparous Goats

No differences were found in any of the behaviors recorded in the nulliparous goats between the two treatments, in each phase of the test (p > 0.05; Figure 3A).

3.4. Comparison Within Treatments in the Maternal Motivation Test in Nulliparous Goats

In group EB2, the average number of low-pitch bleats was higher after than before VCS (p = 0.043; Figure 3A). The frequency of location changes was higher before than after VCS (p = 0.05). Suckling refusals tended to be more frequent after stimulation than before stimulation (p = 0.06). In group P4 + EB2, there were no differences in all behaviors recorded in this treatment when goats were compared before and after VCS (p > 0.05).

3.5. Comparison Between Treatments in the Maternal Motivation Test in Multiparous Goats

The frequency of smelling the kids before VCS was higher in the B2 group than in the P4 + B2 group (p = 0.01; Figure 3B). The frequency of aggression toward the kids before VCS was higher in goats given the EB2 treatment than in those given the P4 + EB2 treatment (p = 0.016). The frequency of high-pitch bleats before VCS tended to be higher in goats given the EB2 treatment than those given the P4 + EB2 treatment (p = 0.081). The time spent cleaning the kids after VCS tended to be longer in EB2-treated goats than in P4 + EB2-treated goats (80.1 ± 67.3 vs. 2.4 ± 2.4 s; p = 0.080).

3.6. Comparison Within Treatments in the Maternal Motivation Test in Multiparous Goats

In group EB2, the frequency of low-pitch bleats was higher after than before VCS (p = 0.012; Figure 3B). The duration of cleaning the kids was longer after than before VCS (80.1 ± 67.3 vs. 0 ± 0 s; p = 0.028). In the P4 + B2 group, the frequency of smelling the kids was higher after than before VCS (p = 0.034). Location changes tended to be more frequent after than before VCS (p = 0.063). The frequency of aggression toward the kid tended to be higher after than before VCS (p = 0.10).

3.7. Maternal Index Comparing Within Treatments Without Considering Parity

Goats given the EB2-only treatment were mostly maternal after vs. before VCS (p = 0.002; Figure 4). Goats given the P4 + EB2 treatment showed no change in the maternal index before vs. after VCS (p = 0.861).

3.8. Maternal Index Comparing Before and After VCS for Each Parity Without Considering Hormonal Treatment

Nulliparous goats, regardless of the induction treatment, showed no difference before compared with after VCS (p = 0.53; Figure 5), while multiparous goats, regardless of the induction treatment, were more maternal after compared with before VCS (p = 0.007).

3.9. Maternal Index of Nulliparous or Multiparous Goats, Comparing Between Treatments, Without Considering Stage of Test (Before or After VCS)

In general, multiparous females had a positive maternal index with both treatments, although no differences were found between the two treatments (p = 0.953). Nulliparous females had a negative maternal index with the EB2 treatment and a positive maternal index with the P4 + EB2 treatment; this difference between treatments was significant (p = 0.045; Figure 6).

4. Discussion

Our hypothesis that we could induce maternal behaviors in ovariectomized goats by administering estradiol benzoate (EB2) or progesterone plus estradiol benzoate (P4 + EB2) was confirmed. Additionally, we demonstrated that this response is potentiated when VCS is performed. Moreover, we demonstrated the hypothesis that maternal experience can affect the response to these treatments.

Our results show that when treatment with EB2 or P4 + EB2 was applied to ovariectomized goats exposed to a newborn kid, they exhibited different behaviors associated with maternity. For example, when these goats received the hormonal treatments, they emitted low-pitch bleats, smelled and cleaned the kid, and allowed it to approach the udders and suckle. These results align with those reported in several studies conducted in goats, where progesterone concentrations decreased and estradiol concentrations increased at the end of gestation [8,33]. They manifested maternal motivation immediately after parturition, as females were attracted to the kid and immediately initiated cleaning, emitting low-pitch bleats, and allowed udder acceptance and suckling [34,35].

Goats treated with both hormones showed negative behaviors toward their offspring, such as udder rejection and aggression. This suggests they can be selective even while demonstrating maternal motivation. Several goat studies report that females behave aggressively and reject alien offspring, especially once maternal selectivity is established [35]. This supports that estradiol and progesterone regulate maternal behavior in goats, starting before birth and continuing after, in line with changes in estrogen and progesterone levels as previously reported [8].

Meanwhile, a study carried out in ovariectomized ewes treated with progesterone or estradiol benzoate plus progesterone found differences in some behaviors associated with maternal motivation, such as cleaning and suckling, acceptance of the offspring, and manifestations of rejection of the offspring [27], which agrees with our results.

Building on these findings, although we observed manifestations of maternal motivation in the presence of a kid in the goats that underwent both treatments, we also found that the females showed certain behavioral differences depending on the treatment. Specifically, goats with EB2 emitted a higher frequency of high-pitch bleats before VCS than goats with EB2 + P4.

Treatment with P4 + EB2 significantly influenced the behavior of multiparous goats, as it reduced the emission of loud bleats, aggressive behavior, and udder refusals before VCS. Although these steroid-stimulated behavioral changes indicate that the treatment alone favored some signs of maternal behavior, the animals were far from being effectively maternal. However negative behavioral responses toward kids were reduced when VCS was applied after the P4 + EB2 treatment, highlighting the interaction between treatment and VCS. The results of aggression, udder refusals, and high-pitch bleating coincide with those reported by Kendrick et al. [11], where the frequencies of aggression, udder refusals, and high-pitch bleating were lower after VCS in nulliparous and multiparous ewes ovariectomized with a treatment with estradiol benzoate or estradiol benzoate plus progesterone. This similarity may be because prior stimulation with both steroids is necessary in goats, as in sheep. Furthermore, the maternal index in multiparous goats was higher after VCS. This response was similar to that reported in ewes with progesterone plus estradiol treatments, where a higher maternal score was observed after VCS [27].

The hormonal treatments used in this study had effects on both nulliparous and primiparous females, which differed from those reported in a previous study in sheep, in which intact females with a short treatment including estradiol and progesterone induced maternal behavior in only 7 of 12 multiparous females, while none of the nulliparous females responded [36]. In our study, treatment with estradiol benzoate generally yielded the best maternal index. Moreover, in contrast to Le Neindre’s study [36], our results showed nulliparous females had a better maternal index with the P4 + EB2 treatment. Maternal experience is a crucial factor that influences offspring survival in small ruminants.

Females with previous births show better skills in caring for their offspring and have a lower risk of mortality, while females giving birth for the first time are affected by dystocic births because they sometimes have very large offspring, and their birth canal is very narrow [24,37,38]. They are also affected by neophobia, which causes them to be stressed and restless, and they may eventually end up abandoning the birthing site [37]. Likewise, depending on their nutritional status at the end of gestation, they may have lower milk production. Therefore, on the farm, it is recommended to pay more attention to females that are about to give birth for the first time, ensure adequate nutrition at the end of gestation, monitor births, attend to dystocia, and be attentive that the female accepts and feeds the newborn. In case of disturbances, this process should be supported.

Temperament is another factor that could interfere with females’ response to hormonal treatments to express maternal behavior. In the literature, most studies on this topic have been carried out in sheep. For example, it was found that calm ewes licked their lambs more and tended to stay longer at the birthing site, while lambs born to nervous mothers got up sooner and began exploring more quickly [39]. It was also shown that maternal behavior and peripartum levels of estradiol and progesterone differed little in Merino sheep selected for their calm or nervous temperament [12]. However, we did not find similar studies in goats, although temperament could affect the display of maternal behavior, as found in sheep [40]. The effect of so-called personality type and its correlations with behavioral and physiological responses has been evaluated [41]. Although various factors that may affect the expression of maternal behavior in small ruminants have been evaluated, none have been identified as facilitating the acceptance of other animals’ offspring, except in exceptional cases, such as adoption processes. This is because these species reproduce seasonally, where births are concentrated at a certain time of year. As such, it is essential to establish a selective bond between mother and offspring quickly to ensure that other offspring will be willing to feed and that the mother devotes her attention exclusively to her own offspring. This type of behavior is crucial to the reproductive success of these species, and natural selection has favored most females that display selective maternal behavior and avoid feeding other alien offspring.

No studies have investigated the role of the mother’s breed in maternal performance in goats, as has been investigated in other species such as sheep and cows. Although previous studies have considered different breeds, such as French Alpine, Toggenburg, Saanen, and Creole [1,26,42,43,44,45], they have either evaluated each breed independently or focused on other factors, rather than the effect of breed. Therefore, it would be interesting to investigate whether there is a breed-derived effect on the expression of maternal behavior, and secondly, whether hormonal treatments, such as those tested in this study, may vary depending on the maternal breed.

The main limitations of our work were the low number of animals in both parities and per treatment. Therefore, the perspectives of this research could include evaluating these hormonal treatments to larger number of animals per parity and per treatment. We could also evaluate whether other factors, such as temperament or the mother’s breed, could have an intersecting effect on the hormonal control of maternal behavior in goats.

5. Conclusions

We concluded that maternal behaviors can be induced in ovariectomized nulliparous and primiparous goats by administering estradiol benzoate (EB2) or progesterone plus estradiol benzoate (P4 + EB2). Regardless of parity, maternal behavior induced with EB2 treatment was better when VCS was applied. Likewise, maternal experience altered the response, with prior maternal experience leading to more pronounced or rapid onset of maternal behaviors.