1.Estrus Signs

The estrus period is categorized in three stages—early, mid, and peak. We collect data on signs and behaviors associated with each of these stages (Table 1). We then classified male/female pandas into each estrus stage based on 10 estrus occurrences during the months from February to June which led to mating in 2001 to 2020 (Table 2). The first day of copulation is designated as Day 0, and dates (mean ± standard deviation) are applied retroactively for the appearance of each sign. The mid and peak estrus periods are in alignment between the males and females, but the early stage is around a month for females while it begins two to three months in advance for males.

• Table 1. Estrus Stage Categorization

  Estrus Stage		Estrus Signs/Behaviors
  Female	Early Estrus	Marking, bathing, genital level 1
  	Mid Estrus	Mating calls, interest in males, genital level 2
  		Increase in vaginal mucosa keratinocytes, rise in estrus hormone levels
  	Peak Estrus	Genital level 3, drop in estrus hormone levels from peak
  		Presenting -> mating
  Male	Early Estrus	Marking, bathing
  	Mid Estrus	Mating calls, interest in females
  	Peak Estrus	Strong interest in females, masturbation -> mating

• Table 2. Estrus Stage Onset

  	Early Estrus	Mid Estrus	Peak Estrus
  Female	Day ‐20.0±12.2	Day ｰ7.6±1.4	Day ｰ0.6±0.8
  Male	ｰ2 to 3 months	Day ｰ6.3±2.0	Day ｰ2.4±1.9

  *First day of copulation is set as Day 0　n=7

Glossary and Definitions

• Genital level： Three-level ranking system based on coloring and swelling
• Mating calls: Unique vocalizations made during estrus
• Vaginal mucosal cells: At this park, cells excreted in the urine are classified into blue nucleated cells, red nucleated cells, and anucleated cells via Papanicolaou test.
• Estrus hormone: At this park, this refers to measurements of Estron-3-Glucuronide (E1G), a urinary estrogen metabolite.

2. Pinpointing Ideal Breeding Seasons

After summarizing estrus signs and E1G changes seen in Rauhin in 2020 (Figure 1), we saw a rise in E1G, an increased number of mating calls, and swelling and redness in the genitals while she approaches peak estrus. After both male and female pandas make mutual calls and the female faces her posterior to the male, raising her tail, the two share a space and ultimately mate. Ovulation in pandas is reportedly to occur when estrus hormone levels drop significantly from peak levels, which means mating in 2020 coincided with ovulation. Babara et al. collected cells from vaginal swabs and classified them into nucleated basophils, nucleated acidophils, and anucleated keratinocytes via Papanicolaou test (in 2003). They reported that approximately a week before mating, basophil and acidophil levels become equal while two to three days before mating the ratio of keratinocytes exceeds that of the other cells, which is effective in predicting the appropriate timing for mating in advance. Here at this park, we collect vaginal mucosal cells from exfoliated cells excreted in urine instead of collecting them directly from vagina in an effort to reduce the stress placed on our animals. Fluctuations in vaginal mucosa in Rauhin (Figure 2) show acidophils growing dominant six days prior to mating and keratinocytes two days prior, the same as results from exfoliated cells in urinary excretions.

Exfoliated Vaginal Mucosal Cells Released in Urine (Papanicolaou Test)

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  Mid Estrus (nucleated basophils and nucleated acidophils)

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  Peak Estrus (predominant anucleated keratinocyte)

Figure 1. Estrus Signs and Fluctuations of E1G in Urine (Rauhin in 2020)

Figure 2. Vaginal Mucosal Cells Released in Urine (Rauhin in 2020)

3. Mating Results

In order to prioritize natural mating, we worked to synchronize estrus stages for a male and a female pandas. Between winter and spring we cycled playgrounds frequently to allow them to smell excrement and markings. As a result, we saw 14 cases of mating from 2001 to 2020. With a priority given to natural mating, we only had three instances of artificial insemination (in 2001, 2006, and 2008), when the female was in estrus but the male showed no sign of interest. Out of these 14 cases 11 births were observed, granting a high conception rate of 78.6%. Today, aging of our breeding individuals is becoming an issue to tackle with. For instance, in the case of the male Eimei, from 2016 (age 24) onward we let him meet with female through cages within a shortest possible time span and granted him at least seven hours of rest between mating sessions in order to conserve his physical strength for the process.

Breeding Performance at Adventure World