Ok, was an overlook on my side. The MTU fix is only applicable on the 64Bit platforms.
I suggest to try SMB3 and see if that helps. Maybe the issue is something totally different. Things you could test:
- Test network on the Pi3 with IPerf3
- Try to play the file locally from USB
Maybe something changed in the network, we suggest to check your network with iperf3. Please read this howto