The Benefi ts of Freezing Embryos and Oocytes for Long-term Storage In laboratory animals, especially the mouse, advances in molecular genetics has permitted the
creation of thousands of mutant mouse strains that are too expensive to maintain as live breeding colonies (1). As a result, an increasing number of genetically valuable mouse strains are
preserved and “maintained” by freezing their embryos, oocytes and sperm. Embryo cryopreservation and transfer have become prominent in livestock industry (especially for large
and small ruminant breeding) (2) to improve the propagation of genetically valuable individuals.
In 2005, more than 370,000 frozen-thawed bovine embryos were transferred worldwide (2). At
last, the benefi ts of cryopreserved oocytes and embryos are widely recognized for wild species
conservation because frozen germ cells and embryos could serve as assurances against any
unforeseen catastrophes and assist in the management of the populations both in zoos and in
nature (3). However, the application of this technology in wildlife conservation is still far from
being routinely used (4). So far, live offspring have been produced from frozen-thawed embryos
only in less than a dozen of species (felids: ocelots, caracal, African wild cat; primates: macaque,
baboon; ungulates: eland, red deer, fallow deer) (4).
Contents Mitochondria play fundamental roles during oocyte development. The accumulation and spatial redistribution of these energy‐producing organelles have been linked to the developmental competence of mammalian oocytes. Here, we assessed the copy number, distribution and activity of mitochondria within cat oocytes during folliculogenesis. In Experiment 1, oocytes were recovered from primordial ( n = 152), primary (112), secondary (95), early (131), small (118), antral (86) and advanced antral (5) stages follicles, and mitochondria DNA extracted and quantified using qPCR . In Experiment 2, oocytes from pre‐antral ( n = 44), early antral ( n = 66), small antral ( n = 59), antral ( n = 41) and advanced antral ( n = 21) follicles were isolated and stained with CMXR os MitoTracker dye to assess mitochondrial distribution pattern and activity levels. Oocyte's mitochondria DNA (mt DNA ) copy numbers gradually increased as folliculogenesis progressed, with a significant shift at the small antral stage (0.5 to <1 mm in diameter). The location of mitochondria gradually shifted from a homogeneous distribution throughout the cytoplasm in pre‐antral oocytes to a pericortical concentration in the advanced antral stage. Quantification of CMXR os fluorescent intensity revealed a progressive increase in mitochondrial activity in oocytes from the pre‐antral to the large antral follicles. Taken together, these findings demonstrated that cat oocytes undergo dynamic changes in mitochondrial copy number, distribution and activity during folliculogenesis. These significant modifications to this crucial cytoplasmic organelle are likely associated with the acquisition of developmental competency by cat oocytes.
Matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) are known to play key roles in the remodelling of extracellular matrix during ovarian folliculogenesis, especially during the final stage of follicle development. To date, little is known about the significance of MMPs and TIMPs during preantral follicle development. This study determined the expression of MMPs and TIMP-1 during various stages of cat folliculogenesis, largely for the purpose of securing information useful to improving in vitro follicle culture. Primordial (~10 follicles/cat), primary (~5 follicles/cat), secondary (~9 follicles/cat), early antral (~9 follicles/cat), and antral (~4 follicles/cat) follicles were physically isolated from ovaries recovered from 15 cats (5 months to 3 years old during follicular stage) undergoing ovariohysterectomy and assessed for expression of MMP-1, -2, -3, -7, -9, and -13 as well as TIMP-1 using real-time quantitative polymerase chain reaction (q-PCR; 2–4 replicates/follicle stage). Additional ovaries were obtained from three prepubertal (6 months old) and three adult (1 year old) cats and ovarian pieces were fixed in Bouin's solution and assessed for MMP-2 and -13 localization using immunohistochemistry. MMP expression data were analysed using the Kruskull-Wallis one-way ANOVA. Follicles from all stages of development expressed MMPs and TIMP-1. Specifically, expression of MMP-2 increased (P < 0.05) as folliculogenesis progressed (10-fold increases from primordial to early-antral and antral stage). There were no differences (P > 0.05) in the expression of other MMPs among follicular classes. For TIMP-1, there was a tendency (P = 0.07) for increased expression after antrum formation (early antral and antral stages). Immunohistochemistry analysis revealed that MMP-2 was expressed in both the oocyte and somatic cells of all follicular stages in prepubertal cats. However, MMP-2 expression was limited to granulosa and theca cells of antral follicles in adult females. MMP-13 was expressed in the granulosa and theca cells of primary, secondary, and antral stage follicles, and there were no differences (P > 0.05) in localization patterns for this protein between prepubertal and adult females. In summary, the study is the first to report the expression of MMPs as well as TIMP-1 in isolated cat follicles. The difference in MMP-2 expression between prepubertal and adult cats suggests that there may be age-specific requirements for in vitro follicle growth. We are keenly interested in this information for underpinning the development of new in vitro microenvironments for growing immature cat follicles. We suspect that such information will be crucial for understanding how to promote the remodelling of the extracellular matrix by creating degradable biomaterials containing MMP-sensitive peptides to allow optimal follicle expansion.
Abstract Anthropogenic change is a major threat to individual species and biodiversity. Yet the behavioral and physiological responses of animals to these changes remain understudied. This is due to the technological challenges in assessing these effects in situ. Using captive maned wolves (Chrysocyon brachyurus, n = 6) as a model, we deployed implantable biologgers and collected physiological data on heart rate (HR) and heart rate variability (HRV) over a 1-year period. To test for links between HR and changes in the environment we analysed HR daily rhythms and responses to potential stressors (e.g. physical restraint, change in housing conditions, short-distance transportation and unfamiliar human presence). The 2-min HR averages ranged from 33 to 250 bpm, with an overall rest average of 73 bpm and a maximum of 296 bpm. On average, HRV was higher in females (227 ± 51 ms) than in males (151 ± 51 ms). As expected, HR increased at dusk and night when animals were more active and in response to stressors. Sudden decreases in HR were observed during transportation in three wolves, suggestive of fear bradycardia. We provide the first non-anesthetic HR values for the species and confirm that behaviour does not always reflect the shifts in autonomic tone in response to perceived threats. Because strong HR responses often were not revealed by observable changes in behaviour, our findings suggest that the number and variety of stressors in ex situ or in situ environments for maned wolves and most wildlife species may be underestimated. Our study also shows that integrating biologging with behavioral observations can provide vital information to guide captive management. Similar technology can be used to advance in situ research for developing more effective welfare, management and conservation plans for the species.
Summary Animals moving through landscapes need to strike a balance between finding sufficient resources to grow and reproduce while minimizing encounters with predators 1,2 . Because encounter rates are determined by the average distance over which directed motion persists 1,3–5 , this trade-off should be apparent in individuals’ movement. Using GPS data from 1,396 individuals across 62 species of terrestrial mammals, we show how predators maintained directed motion ~7 times longer than for similarly-sized prey, revealing how prey species must trade off search efficiency against predator encounter rates. Individual search strategies were also modulated by resource abundance, with prey species forced to risk higher predator encounter rates when resources were scarce. These findings highlight the interplay between encounter rates and resource availability in shaping broad patterns mammalian movement strategies.
Understanding stage-specific requirements of mammalian folliculogenesis is limited in the domestic dog. The present study examined the effects of two potential regulators of dog follicle growth and survival in vitro, namely the original stage of the follicle (i.e. preantral (≤230µm diameter) vs early antral (diameter from >230 to ≤330µm) and FSH and/or LH concentrations. After isolation and alginate encapsulation, follicles were cultured in 0, 1, 10 or 100µgmL-1 FSH and 0, 1 or 10ngmL-1 LH for 20 days. Regardless of stage, FSH promoted growth, but LH did the same only in the absence of FSH. Production of 17β-oestradiol and progesterone was detectable, indicating theca cell activity. The greatest growth occurred in preantral (mean (± s.d.) 61.4±25.9%) versus antral (42.6±20.3%) follicles, but neither developmental stage nor gonadotropin affected survival. Antrum detection was minimal due, in part, to antral collapse, and oocytes exhibited an increasingly pale appearance and chromatin degeneration over time. The results demonstrate that pre- and early antral stage dog follicles encapsulated in alginate grow significantly in vitro. However, because FSH and LH alone or in combination fail to promote antrum development, the next step is identifying factors that enhance antral expansion.
