Thesis

Investigation of the mode of ovulation and ovarian cycle in bears

Literature focusing on the reproductive mechanisms in bears is often limited and only has a narrow scope. However, with six out of eight bear species being considered threatened or endangered, it seems critical that more information about reproduction be obtained through empirical studies. One of the reasons that little is known about reproduction in bears is due to a variety of reproductive mechanisms that bears have evolved that make studying reproduction difficult. This in addition to their solitary nature, makes long-term studies of wild bears challenging. These reproductive mechanisms include: induced ovulation, pseudopregnancy, delayed implantation (embryonic diapause), seasonal breeding, multiple paternity, and polyestrus (with subsequent ovulation events). The current thesis focuses on two separate, but related, projects that I was involved in, focusing on reproduction in American black bears (Ursus americanus) and the polar bear (Ursus maritimus). In the first portion of my thesis, I re-examine the ovulation mechanism in American black bears, and in the second portion of my thesis, I work to determine whether a captive female polar bear had ovarian activity. In bear biology, it has been accepted that American black bears are coitus-induced ovulators, although there is little empirical evidence that supports this. Induced ovulation is a process in which ovulation is caused by an external stimulus. In Chapter 2, we work to determine whether coitus is the only stimulus that induces ovulation in American black bears. We did this using physiological measures of estrus (vulva score), steroid analyses on urine and serum samples during embryonic diapause, and histology of ovarian structures. All of these measures serve as proxies for or direct evidence of ovulation. Females were categorized into three groups: mated, not observed mating, and isolated. These groups were compared to detect differences in estrus length, length of time it takes to exit estrus after mating, progesterone (P4) concentrations during embryonic diapause and estrogen (E2) patterns during estrus. In the current study, we found that the length of estrus and the time it takes to exit estrus were not statistically different between the female groups. Likewise, we found that the concentrations of P4 during both early and late embryonic diapause were equally elevated. We also found that isolated females exhibited E2 patterns indicative of ovulation. Finally, we confirmed that two of our females that were not observed mating had CL present on their ovaries via histological analysis. From this, we were able to conclude that American black bears are ovulating due to a stimulus other than coitus. We were unable to determine what stimulus, if any, induces ovulation in or if American black bears are spontaneous ovulators due to limitations in study design and the field site. Similar to American black bears, polar bears are also considered coitus-induced ovulators although there is little empirical evidence supporting this. Because polar bears are considered coitus-induced ovulators, it would be expected that a mature female, housed singly, without any male stimuli would exhibit no signs of pseudopregnancy or ovulation due to no possibility for coital or other stimulation. Although this prediction agrees with the current literature, zoo keepers at the Milwaukee County (MKE) Zoo reported that their 30 year old female was exhibiting the behavioral signs of pregnancy. As result, the MKE Zoo asked Dr. Spady to look into ways to determine whether their female was still cycling. Because P4 is associated with pregnancy and pseudopregnancy, and is known to increase after fertilization and again at implantation, we decided to use this as our measure for ovarian activity. Keepers at the MKE Zoo collected fecal samples whenever possible for four years and then shipped them to CSUSM. In the lab at CSUSM, we used liquid phase extraction to separate the steroid metabolites from the fecal samples. We then measured the concentrations of progestins (P) in the samples via an EIA. We found that in 2010 and 2014, the female from the MKE Zoo exhibited P patterns that match what would be expected in a pregnant/pseudopregnant bear. In 2011, we determined that the female was not cycling and likely exhibited a luteal cyst which would have caused elevated P concentrations at times that would otherwise not be expected. In 2015, we also suspected that this female exhibited a luteal cyst due to elevated P concentrations early in the year, but because we do not have samples from later in the year, we were unable to determine if this female exhibited an active luteal phase and was therefore cycling. Because we were able to observe an active luteal phase in this female, we can infer that this female also likely ovulated. Because this female was isolated throughout the study, then it is likely that, like in American black bears, polar bears are not only coitus-induced ovulators. Knowledge obtained through the work of this thesis can be used to better inform both captive and wild management. The fact that ovulation may not require coitus to occur in both American black bears and polar bears means that ovulation and pseudopregnancy are occurring at higher rates than currently expected. If this is true, this knowledge can be used to better inform methods for pregnancy determination. Furthermore, this knowledge can better inform management of captive populations, for example, the reduction of invasive ovulation induction techniques during artificial insemination procedures. There is still much to learn in regards to reproduction in bears, but this thesis reopens issues that may have been misleading researchers in previous years.

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