Trout sperm 100X
María de los Ángeles Peralta-Martínez1*, Salvador Romo García2, Michael E. Kjelland3 and Humberto González-Márquez4 1Posgrado
en Ciencias Biológicas y de la Salud, Universidad Autónoma Metropolitana, Iztapalapa *Email:
[email protected] 2Laboratorio de Reproducción, Departamento de Ciencias Pecuarias, Facultad de Estudios Superiores Cuautitlán, UNAM 3Conservation, Genetics & Biotech, USA 4Departamento de Ciencias de la Salud, Universidad Autónoma Metropolitana Iztapalapa
Introduction The main function of trout semen extenders is to preserve viability while keeping the sperm motility inactivated. The longer the sperm can remain viable and inactivated the better, until being intentionally activated so the sperm can travel to the egg micropyle for fertilization. In Rainbow trout (Oncorhynchus mykiss) considerable advances have been made concerning the production of sperm extenders, protocols and handling of the gametes. However, the protocols for using sperm extender can differ considerably with regard to composition of the extenders. Additionally, the precise impact of pH on sperm motility and its effect on the design and selection of sperm extender media has not yet been fully established. The objective of this study was to determine the effect of pH of 5 extenders on Rainbow trout sperm motility and activation during storage at 5°C.
9
9
8
8
7
7
6
6
pH
pH
a)
5
5
4
4
3
3
2
2 0
1
2
306
512
5 6 Days ERDHAL HANKS
7
b)
0
1
306
512
8
MOUNIBS
2
5 6 Days ERDHAL HANKS
7
8
MOUNIBS
Figure 1. Buffer capacity of a) unsterilized extenders and b) sterilized extenders.
Methods
a)
Two year-old sexually mature Rainbow trout males (n=46) weighing 1.5 to 2.5 kg were caught at Aquaculture Farm Tatakay in Jilotzingo, Estado de México, México. One semen sample per male was collected by abdominal massage.
100
Motility (%)
80 60
306 HANKS MOUNIBS ERDHAL
40 20 512
0 7
7.2
7.4
7.8
8
8.2
pH
Trout: Artyom Yefimov/123RF
Results 100
To evaluate pH dynamics in each extender, pH was tested daily over an 8-d storage interval. Trout sperm were added to each of the extenders in a ratio of 1:1 and motility recorded. Afterwards, each activator solution (i.e., DIA 532, saline solution (0.85%) and pond water) was added separately to a sample of the extended sperm, to initiate sperm activation. Motility was evaluated subjectively at 400x and monitored until ~99% of the sperm stopped moving (on average, in 40 s). During this storage period all of the samples were refrigerated at 5 °C.
Extenders kept their buffer capacity through time if they were sterilized and not contaminated with bacteria, which can acidify the medium (Figure 1). Extender 512 activated motility when pH was higher than 7.4. By contrast, extender 306 activated motility at every pH. Hanks solution activated motility at very low percentages with the different pH levels. Mounib, and Erdahal and Graham solutions did not activate motility for any pH tested (Figure 2).
80
Conclusions
EF HANKS MOUNIBS ERDAHL 512 306
40
0 7
7.4
7.8
8
8.2
c)
100 80 60
EF HANKS MOUN… ERDAHL 512 306
40 20
0 7
Extender 512 produced the best sperm storage results. Sterilization of storage solutions is recommended to avoid bacteria contamination that can acidify the medium. The pH of the extender solutions affected sperm motility, however, further studies are needed to determine the mechanism of action.
7.2
pH
Extender 512 presented the best (p
