A-LevelBiologyGenetic technology9700/53 · May/June 2019 · Q210 Marks

For many years, farmers have used artificial insemination (AI) as a method to improve the milk yield of dairy cattle. In cattle, sperm cells carrying the X chromosome contain 3.8% more DNA than the sperm cells carrying the Y chromosome. Female cattle have the sex chromosomes XX. Assisted reproduction programmes allow dairy farmers to increase the number of female offspring produced. One technique used to identify which sex chromosome a sperm cell contains is centrifugation. Some semen samples were collected from male cattle. • 10 cm³ samples of semen were placed in centrifuge tubes with 15 cm³ of a buffer solution added to each tube. • The tubes were placed in a centrifuge and left to spin for 20 minutes at 1275 revolutions per minute. • The spinning causes the sperm cells containing the X chromosome to fall to the bottom of the tubes. The sperm cells containing the Y chromosome remain in the fluid at the top. Semen samples which have been processed in this way are referred to as sexed samples. Another method of sexing semen samples is to use a technique called flow cytometry as shown in Fig. 2.1. [Figure 2.1] The samples containing X chromosomes are frozen in small plastic straws, allowing semen samples to be stored for many years and transported all over the world. • Flow cytometry can process 8000 sperm cells every second. • One straw can hold 2.0 × 10⁸ sperm cells. The semen is analysed by technicians to determine its quality before it is sexed. Technicians use microscopy to look at the appearance and motility of the sperm cells and to estimate the sperm count. Fig. 2.2 shows the surface view of a haemocytometer used to estimate the number of sperm cells in a sample. The depth of the haemocytometer is 0.1 mm. [Figure 2.2]

(a)(i)State three variables which were standardised in this technique.[3]
(a)(ii)Suggest one reason why it is important that the semen sample is not spun at a very high speed.[1]
(b)Calculate how many straws can be filled every hour by flow cytometry. Number of straws filled every hour =[1]
(c)Suggest how this apparatus could be used to estimate the number of sperm cells per cm³ of semen and describe how the technician decided which sperm cells to include in the count.[5]

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Mark scheme

Marking points from the official mark scheme (May/June 2019). The mark or mark type is shown in brackets.

[3] any 3 of: 1. (same) volume / 10 cm³, of semen ; 2. (same) speed of spinning / spun at 1275 revolutions per minute ; 3. (same) time for spinning / spun for 20 minutes ; 4. (same) volume / 15 cm³, of buffer ;
[1] any 1 of: idea that: some sperm cells may get damaged ; separation of X-bearing and Y-bearing sperm is not achieved / all sperm collect at the bottom of the tube ; AVP;
[1] 14 ;
[5] any 5 of: estimate of numbers: 1. idea of find the volume of the sample or (volume of sample is) (for mm³) 0.25 × 0.25 × 0.1 / 0.00625 / 6.25 × 10-3 (for cm³) 0.025 × 0.025 × 0.01 / 0.00000625 / 6.25 × 10–6 ; 2. divide the number of sperm by the volume or divide 1 mm³ by the volume and multiply by the number of sperm counted ; 3. evidence of correct method of converting to cm³; deciding which sperm to include: 4. idea of: count those where sperm (fully) inside, the square / 0.0625 (mm²) / all (small) squares / haemocytometer/ quadrant/grid / boxes ; 5. idea of: including those in or partially in the inner double lines ; 6. idea of: including those (in edge region) top and left / up and right or exclude those in edge region) bottom and right/down and left ; 7. AVP detail of method: e.g. ref. to, dilution / stirring / even spreading / immobilising sperm / use of coverslip / screenshot for counting / idea of only counting non- deformed / sperm;

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