Effect of weaning age on morphological and histological changes that occur in a rat’s testis

Background: Weaning of mammalian progeny is associated with a change in body shape and physical characteristics. It programs growth, body composition, and the tempo of physiological development and maturation, as well as litter size and parity and, thereby, reproductive strategy. Methods: To evaluate the effects of weaning age on morphological and histological changes that occur in a rats’ testis, we assessed testis size weekly and histomorphometry in 30 days old pups, and 90 days old mature rats that had been weaned early (d16), normally (d21), or late (d26). Results: Early weaning resulted in large testes at age 30 days and beyond, a wide seminiferous tubule wall, and the ratio of blood vessel to the stroma was bigger than in late weaned animals. At age 90 days, and the next generation the litter size of early-weaned rats was bigger than those weaned on days 26 or 21. Conclusions: Early weaning signifi cantly enhances the testes’ size, vasculature, and seminiferous tubular walls, and enhances the liter size compared to late weaning. Research article Effect of weaning age on morphological and histological changes that occur in a rat’s testis Yonatan Crispel1,2*, Yonatan Danin2, Geula Klorin2,3 and Ze’ev Hochberg2 1Hematological Institute, Rambam Health Care Campus, Haifa, Israel 2Rappaport Family Faculty of Medicine, Technion, Israel Institute of Technology, Haifa, Israel 3Department of Pathology, Rambam Health Care Campus, Haifa, Israel Received: 22 February, 2021 Accepted: 19 March, 2021 Published: 23 March, 2021 *Corresponding author: Yonatan Crispel, Hematological Institute, Rambam Health Care Campus, POB 9602, Haifa 31096, Israel, Tel: +972-4-7771116; E-mail:


Introduction
Mammalian life history starts with a stage of infancy, defi ned mostly by lactation, and subsequently a gradual weaning in humans towards childhood, adolescence, and adult diet [1]. Weaning from lactation is responsive to sex, stress and other environmental cues that are presumed to inform the developing organism about risks and opportunities in its current and future environment [2].
The weaning age defi nes life history transition from infancy to childhood in humans and to juvenility in the rat [3] and this transition phase is highly important in shaping (programming for) future growth and maturation trajectories [4].
We have previously shown that in humans a delayed infancy-childhood transition has a lifelong impact [3,4].
Correspondingly, in the rat, the age at weaning from lactation determines a rat's growth trajectory, but also body composition and maturational tempo [5].
We showed that early-weaned rats developed faster than normal, and at adulthood early weaned rats were leaner and longer than late-weaned animals, which were heavier and shorter [5]. Early-weaned progeny matured more rapidly, females showed earlier vaginal opening and estrous and males showed earlier onset of testicular growth [5]. These effects intensifi ed in future generations who were also weaned according to the same schedule.
Surprisingly, early weaned rats and their offspring had bigger testes at maturity [5]; an observation which triggered the present study. Here, we examined the impact of weaning

Short-term effects
To assess the short-term effects and to provide insights about how early weaning alters normal reproductive strategy, we measured testicular size weekly for 12 weeks. Nine males of the early-weaned group (16-day breastfeeding after birth) and nine males of the late-weaned group (26 day) were sacrifi ced.
In future generation, the rat testes were excised on day 30.
To evaluate the long-term consequences of early and late weaning and the effect on testicular size, testicular size was determined daily from day 30 once a week until day 90 using a self-built orchidometer based on the human Prader orchidometer with mock-ups ranging from 0.5 to 5ml [7].
Testes measuring more than 1 ml were considered early pubertal, in fi rst generation second group of rats that had been weaned by cross-foster mothers on d16, or d26 and were sacrifi ced on d90 for testicular histomorphometry.

Histomorphometry
Histologic sections were prepared from 5-6 sites along the testicles, fi xed in 0.05 mmol/L saline phosphate buffer containing 4% formaldehyde. Paraffi n embedded specimens were longitudinally sectioned (6 μm), stained with hematoxylin and eosin for quantitative evaluation of lumen, seminiferous tubule wall, stroma and blood vessels, Figure 1.
An Image analyzer (Trichip RGB video camera; Sony, Tokyo, Japan) installed on a light microscope (Zeiss, Jena, Germany) and attached to a computer equipped with a frame grabber was used to analyze the extension of positively stained tissue, as previously described [8,9]. Images were captured, digitized, and displayed on a high-resolution colored monitor. The ten most intensely stained fi elds were analyzed at a power lens of 20×10. Images were loaded on screen buffers having a resolution of 760×570 pixels and measured in standardized frames (62993 μm 2 ). Image Pro Plus 4 software (Media Cybernetics, Baltimore, MD) was used to assess the extension of positively stained cells/tissue after segmentation and thresholding.

Statistical analysis
Physiological data were analyzed using SAS 6.12, utilizing two-way ANOVA. Histomorphometric data were analyzed using SPSS 6.0 (Chicago, IL). Positively stained cases in two groups were compared by the Mann-Whitney U test. Comparison between cases used the nonparametric Kruskal-Wallis ANOVA followed by a corrected Mann-Whitney U test for multiple comparisons. Differences with 2-tailed P values < 0.05 were considered signifi cant.

Results
The early-weaned animals' day 16 had bigger testes than late-weaned animals' day 26.

Discussion
This study is part of a previous study examining the effect of weaning from lactation on rat growth, in main study we found that early-weaned rat have a large testis signifi cantly compared late weaning. To explore the reason of this diversity, Histologic and morphologic examine were taken to fi nd differences between testicles of early and late weaned rats. Two central differences between the testicles of the different groups at histological level found: many blood vessels with a thinner wall and a larger lumen compared to animals weaned later. Recent data suggests that Sertoli cells play a key role with vasculature network formation [10], the vascular density of a tissue is infl uenced by diverse factors, both pathologic and physiologic.
For example, it is well established that angiogenesis is a part of     pathologic malignant processes [11,12] increased angiogenesis, blood vessel concentration can be a favorable process in which growth, and development is achieved in breastfeeding, wound healing [13] Or infant organ development, this fi nding infl uence metabolic states and we assume that this result derive from different Sertoli concentration with different breastfeeding duration, Which can explain the expression of the large testis volume and explain larger litter in early-weaned group as expected from the results.

Lumen wall ratio W a l l -t u b u l e ratio S t r o m a -t u b u l e ratio
The age at sexual maturation plays a central role in reproductive strategy. We thus predicted and found that earlyweaned females showed earlier vaginal opening and estrous, and early-weaned males showed earlier onset of testicular growth and attainment of greater testicular volume compared to late-weaned rats [5]. We previously showed that earlyweaned progeny is more productive than the progeny of laterweaned rats.
It was previously shown that the critical period for testicular development in the rat is from day 13 of gestation to the 21st postnatal day, and that this stage determines the tissue future structure and function [14]. Here, we focused on a developmental milestone at day 21 -weaning from lactation and its effect on the testes and reproduction.
In the wild, weaning from lactation is responsive to stress and other environmental cues that are presumed to program the developing organism for risks and opportunities in its current and future environment [2]. In the animals facility, the milestone of weaning is determined by the facility's routine, and it is customary to separate mothers from offspring on day 21, and this day is defi ned as the life history transition from infancy to juvenility in the rat [3]. This transition is important in programming for future growth and maturation trajectories [4].
The fi ndings presented here on rats lend support to the proposition that the duration of infancy, as indexed by weaning age, predicts and perhaps programs testicular growth, litter size and parity, and, thereby, reproductive strategy.
We have previously shown that short infancy (early weaning) is associated with characteristic fast reproduction long body, underweight, glucose tolerance and insulin sensitivity, but also in large testes and large liter [7]. Among the smallest mammals, testes size is associated with high copulatory frequency and sperm production [15], as observed also in the rats of the present study.
Here, we show the testicles' structures in early-weaned animals that support greater fertility. These structural components include greater seminiferous tubular volumes, seminiferous tubular wall thickness (as a surrogate for Sertoli cells) and more vasculature in the stroma.
In another rodent -the golden hamster, an active testicular tissue is comprised of 92.5% seminiferous tubules and 1.4% of Leydig cell stroma [16].

Conclusion
Seminiferous tubular wall thickness / Sertoli cells support enhanced reproductive capacity in animal that were weaned early. We propose that this phenomenon is an evolutionary strategy to enhance reproduction in animal with a fast life history. Despite substantial heritability in testicular development, much variation remains to be explained, leaving room for the infl uence of environmental factors to adaptively adjust the phenotype in the service of fi tness goals.