Effects of Embryo Transfer on Emotional Behaviors in C57BL/6 Mice

Abstract

Microbiologic standardization plays a key role in the management of animal facilities because contamination of stock could affect the health status and wellbeing of animals and thereby induce artifacts in biomedical research. One common method to avoid the dissemination of pathogens is embryo transfer (ET). Although disturbances in the perinatal environment may cause long-lasting effects on the behavior and physiology of mouse offspring, the influences of ET during this sensitive phase have not yet been addressed. Our study investigated the effects of various components of ET (anesthesia, surgery, recipient strain) on the behavior of dams (exploration, nest-building) and offspring (nest-building, exploration, anxiety, and social and depressive-like behaviors). For ET, the donor strain C57BL/6N and a standard protocol were used. Whereas treatment with anesthesia-analgesia did not affect maternal behavior, female offspring demonstrated overall effects on weight gain and corticosterone levels. Compared with naturally delivered female offspring, dams obtained through ET demonstrated decreased exploration and nest-building. In addition, female ET-derived offspring had enhanced levels of anxiety and increased social interest. Furthermore, ET-derived dams obtained by using NMRI as the recipient strain showed increased exploratory behavior compared with that of dams obtained by using C57 mice as recipients. Compared with using C57 as recipients, both sexes of offspring transferred into NMRI recipients weighed more, and female mice showed a depressive-like phenotype. Our findings suggest that ET, now considered to be a routine procedure in animal husbandry, bears the risk of introducing artifacts.

Figure 1.

Overview. (A) Preliminary experiment: timeline and comparison between intact and sham-operated dams and their respective litters. (B) Timeline and comparison between control (natural conception) and ET dams and their respective littermates and between C57BL/6 and NMRI pseudopregnant recipients of ET embryos and their respective litters.

Figure 2.

Maternal behavior. (A) Perinatal exploration of ET and control dams in the novel-cage test. Control mothers exhibited longer latencies to explore a novel cage during pregnancy but higher levels of exploration during the nursing period. (B) Perinatal nest-building performance of ET and control dams in the novel-cage test. In the 2nd week of pregnancy, control mothers demonstrated increased nest scores, compared with ET dams. In the 3rd week, control dams showed decreased nest-building performance. (C) Perinatal exploration in ET-C57 and ET-NMRI dams in the novel-cage test. Recipient mothers of the NMRI strain exhibited increased rearing behavior in the 3rd week of pregnancy and 2nd week of nursing. In C57BL/6 dams, the latency to first rearing was decreased during the 2nd week of nursing. *, P < 0.05; †, P < 0.01.

Figure 3.

Effect of ET on offspring. (A) Weight development of offspring reared by control and ET mothers. No significant differences occurred between the groups. (B) Anxiety behavior in the O-maze test of offspring of control and ET mothers. Control female progeny had more exits than did female offspring of ET mothers. (C) Social behavior of control and ET offspring during phase 3 of the social recognition test. Female offspring of ET dams demonstrated more entries into the compartment containing an unfamiliar mouse. (D) Social behavior of control and ET offspring during phase 4. Again, female progeny of ET mothers had more entries into the compartment containing the previously unfamiliar mouse than did control female offspring. *, P < 0.05.

Figure 4.

Effect of recipient strain on ET progeny. (A) Weight development of offspring raised by C57 or NMRI recipients. Male and female offspring of ET-NMRI dams weighed more than did those from ET-C57 dams. B) Depressive-like behavior in the forced-swim test. Female mice in the ET-NMRI group had higher levels of immobility on day 1 than did female progeny in the ET-C57 group. (C) Depressive-like behavior in the forced-swim test. Female progeny nursed by ET-NMRI dams displayed longer latency until the first immobility than did female mice nursed by ET-C57 dams. *, P < 0.05; †, P < 0.01; ‡, P < 0.001.